Process for the production of temperature-induced tumor cell lysates for use as immunogenic compounds

ABSTRACT

The present invention relates to a process for the production of an immunogenic compound comprising inducing necrosis by temperature in tumor cells and lysing said necrotic tumor cells so as to obtain a lysate. Furthermore, the invention provides a method for the production of a pharmaceutical composition. Additionally, the invention relates to a pharmaceutical composition comprising a lysate obtainable by the aforementioned process. Moreover, methods and uses for vaccination against cancers, tumorous diseases, infections and/or autoimmune diseases comprising administering the cell lysates of the invention or dendritic cells loaded with the cell lysate loaded to an individual are provided.

The present invention relates to a process for the production of animmunogenic compound comprising inducing necrosis by temperature intumor cells and lysing said necrotic tumor cells so as to obtain alysate. Furthermore, the invention provides a method for the productionof a pharmaceutical composition. Additionally, the invention relates toa pharmaceutical composition comprising a lysate obtainable by theaforementioned process. Moreover, methods and uses for vaccinationagainst cancers, tumorous diseases, infections and/or autoimmunediseases comprising administering the cell lysates of the invention ordendritic cells loaded with the cell lysate loaded to an individual areprovided.

Vaccination involves the administration of an agent to an individual,which will stimulate the immune system to react against the “foreign”components of the vaccine. The vaccine can be administered, inter alia,into the skin, muscle, intraorally, subcutaneous, intradermally,intranodally, intraperitoneally, intra- or peritumorally orintravenously. The foreign components of the vaccine are known as“antigens”. As a result of a vaccination procedure an individualdevelops immunity so that a subsequent exposure to the antigen(s) willevoke a response to eliminate or destroy the antigen carrying cells,organisms or particles or improve the disease symptoms or protect from aconnected disease. Vaccines have been highly effective in protectingpeople from infectious organisms. Vaccinations for bacterial and viralinfectious agents are now routinely used for: influenza viruses,measles, chicken pox, polio, pneumococcal bacteria, and hepatitisviruses and the like.

Because of the success in immunizing individuals against certaininfectious organisms, it has been a great task of clinicians andscientists to develop effective vaccines against cancers.

The fight against infectious diseases with vaccines also teaches thatprevention of infectious diseases with vaccines is easier than therapyof the same disease under development. This experience has beeninterpreted as suggesting that prophylactic vaccination against cancermay be more successful than vaccination when the disease is at anadvanced stage. Therefore, several different types of cancer vaccines orimmune therapies are under development and are aimed to be used forpreventing, ameliorating and/or treating cancer and/or tumorousdiseases.

In principle, the assumption of the foregoing was the following: tumorcells can be weakened, or attenuated, and injected like a vaccine into amammal, e.g. a mouse. Afterwards, if these same tumor cells, at fullstrength, are injected into the mouse, the mouse will reject or fightthe tumor cells and cancer will not develop or decrease the tumorbourdon. However, if a mouse has not been vaccinated, it will developcancer.

Immuntherapies for preventing, ameliorating and/or treating cancer andtumorous diseases by means of using whole cell vaccines (WCV) have theadvantage of being multivalent with respect to tumor-antigens, howeverWCVs are often only weakly immunogenic. In general, it is to bedistinguished between vaccines comprising vital whole tumor cells (WCV)and vaccines comprising lysed tumor cells (TCLV) (Sivanandham (2000),Biological Therapy of cancer, Ed. Rosenberg, S. A., 632-647).

Whole cell vaccines (WCV) have the advantage that tumor cells can begenetically engineered before being vaccinated and, therefore, used as avehicle for substances that have an immunstimulatory effect (Mach(2000), Curr. Opin. Immunol. 12, 571-575). However, the disadvantages ofthis therapy are the high technical expense of genetically engineeringsaid cells, the problems of keeping a high quality standard in theproduction of whole cell vaccines and the ethical problems accompanyingthe administration of vital tumor cells as vaccines (Sivanandham (2000),loc. cit.).

With respect to WCV, several publications showed that the kind of celldeath influences the immune response (Melcher (1999), J. Mol. Med. 77,824-833). Additionally, it was found that the expression of heat shockproteins influences the immune response against cells (Galucci (2001),Curr. Opin. Immunol. 13, 114-119; Todryk (2000), Immunology 163,1398-1408). Moreover, a large number of methods to induce cell-death, inparticular, apoptosis, exists which strongly influence theimmunogenicity of apoptotic tumor cells (Restifo (2000), Curr. Opin.Immunol. 12, 597-603). With respect to WCV various publications showedthat vital necrotic tumor cells are both in vitro and in vivo, i.e. inthe animal model system, more immunogenic than apoptotic or untreatedcells. In those publications cell death was chemically induced bygancyclovir in tumor cells, which express the herpes simplex virus (HSV)thymidine kinase (HSVtk). Said HSV thymidine kinase functions as aso-called “suicide-gene” if for example induced by gancyclovir. However,cell death only occurs after applying gancyclovir if the cells carryingthe recombinant HSV thymidine kinase gene, additionally harbour theanti-apoptotic gene bcl-2. Whereas HSV thymidine kinase positive andbcl-2 negative cells undergo programmed cell death after induction ofHSV thymidine kinase by gancyclovir. Only the use of HSVtk and bcl-2positive whole tumor cells for prophylactic vaccination of mice and thesubsequent administration of gancyclovir resulted in a protection of theanimals against the development of tumors (Gough (2001), Cancer Res. 61,7240-7247; Melcher (1998), Nat. Med. 4, 581-587). It was also observedthat macrophages phagocytosed both necrotic and apoptotic tumor cells,however, reacted differently with respect to said tumor cells. On onehand immunstimulatory cytokines, like TNFα or IL-1β were secreted if themacrophages phagocytosed necrotic cells, on the other handimmune-suppressive cytokines, like IL-10 were secreted if apoptoticcells were phagocytosed (Gough (2001), loc. cit.). Moreover, macrophageswhich were co-cultivated either with tumor cells incubated for 1 hour at45° C. or tumor cells incubated for 15 minutes at 45° C. were tested fortheir cytokine secretion pattern. If challenged with tumor cells, whichwere heat-induced for 1 hour, the macrophages reacted similarly likethose challenged with chemically induced necrotic cells with respect tothe secretion of cytokines. Whereas tumor cells which were heat-inducedfor 15 minutes resembled apoptotic cells in that they caused secretionof immune-suppressive cytokines after being phagocytosed by macrophages(Gough (2001), loc. cit.).

In contrast to the above-mentioned findings, it was found that livingapoptotic tumor cells are as immunogenic as necrotic tumor cells or evenmore immunogenic (Kotera (2001), Cancer Res. 61, 8105-8109; Restifo(2000), loc. cit.; Shaif-Muthana (2000), Cancer Res. 60, 6441-6447);Albert (1998), Nature 392, 86-89). In these publications other methodsthan those used in the above-mentioned publication for induction of celldeath were performed. For example, Kotera (2001) (loc. cit.) could notfind a difference between allegedly necrotic cells, which were generatedby freezing and thawing and apoptotic cells, which were generated byUV-B treatment when analysing the activation of dendritic cells. Thesame was observed in an animal model system when investigating theprophylactic and therapeutic efficacy of dendritic cells, which hadtaken up either the allegedly necrotic, or apoptotic cells.Shaif-Muthana (2000) (loc. cit.) compared the immunogenicity ofradioactive irradiated apoptotic cells with allegedly necrotic cellsincubated for 30 minutes at 50° C. It was shown that only apoptoticcells could activate T-cells after dendritic cells had phagocytosedthem. Restifo (2000) (loc. cit.) pointed out that it depends on themethod for induction of apoptosis to generate immunogenic apoptoticcells. In particular, apoptotic cells caused by viral infection showedup to be highly immunogenic.

In summary, in view of the above discussed it appears as if differentparameters of whole cell vaccines influence immunogenicity. Inparticular, whole cell vaccines may arise from apoptotic or necroticcells and, thus, may have either stimulatory or suppressive effects onthe cells of the immune system. Additionally, the efficacy of whole cellvaccines on components of the immune system, in particular, macrophages,dendritic cells or T-cells also varies. Another variable parameter,which seems to influence the immunogenicity of whole cell vaccines, isthe kind of technique to induce cell death.

Tumor cells have also been treated with dinitrophenol or fixed withglutaraldehyde in order to improve the immunogenicity before being usedas whole cell vaccines (Berd (1997), J. Clin. Oncol. 15, 2359-2370;Sensi (1997), Clin. Invest. 99, 710-717; Fujiwara (1984), J. Immunol.133, 509-514; Price (1979), Br. J. Cancer 40, 663-665). Additionally,tumor cells have also been treated by physical means, i.e. applying themto high pressure before administration as whole cell vaccines (U.S. Pat.No. 4,931,275). It is of note that these strategies for improvingimmunogenicity may be problematic in that the used chemicals have to beremoved without leaving any residues.

A different approach for treating cancer is hyperthermia. Thereby, tumortissue is treated with heat. This heat-treatment by is based on thefinding that tumor cells are more sensitive to heat than normal cells(Cavaliere (1967), Cancer 20, 1351-1381; Dickson (1979), Lancet 1,202-205). The aim of many workers in the field of hyperthermia was todemonstrate that tumor cells could be killed in vivo and in vitro byheat. Thus, vital tumor cells were treated with temperatures rangingfrom 39° C. to 46° C. to analyse their vitality after the treatmenteither in cell culture or in an animal model system (Cavaliere (1967),loc. cit.; Giovanella (1970), Cancer Res. 6, 1623-1631; Selawry (1957),Cancer Res. 17, 785-791). Besides the vitality of treated tumor cells,also the immunogenicity of said cells was analysed (Bourdon (1981), Ann.Immunol. 1, 43-63; Mise (1990), Cancer Res. 50, 6199-6202; Check (1974),Cancer 34, 197-203; Mondovi (1972), Cancer, 4 885-888; Price (1979),loc. cit.). Mise (1990) (loc. cit.) has isolated cytotoxic T-cells frommice to which tumor cells have been administered and has analysed theability of T-cells to lyse tumor cells in vitro. In these experiments itwas observed that tumor cells, which had been incubated at 42° C. for 30minutes, were more efficiently lysed than untreated tumor cells. Otherstudies could also show the beneficial effect of hyperthermia on thelysis of tumor cells by cells of the immune system.

Clinically hyperthermia is used by applying an increased temperature tothe tumor in vivo in order to achieve a killing of the cells in vivo.Therefore, during a hyperthermia treatment a patient is not immunizedwith tumor cell derived vaccines.

Another approach for prophylactic and therapeutic vaccination againsttumors are tumor cell lysate vaccines (TCLV). The advantages of tumorcell lysate vaccines over the aforementioned approaches for vaccinationand treatment of cancer are the simple method of production, which isnot or much less subjected to fluctuations in keeping a high qualitystandard since tumor cells are killed or lysed before administration.Said killing or lysing can be done mechanically or by freezing/thawing.Additionally, bacterial or viral adjuvants, e.g. Calmette-Guerin (BCG)(Mitchell (1988), Cancer Res. 48, 5883-93) or vaccinia virus(Berthier-Vergnes (1994), Cancer Res. 54, 2433-2439) have been describedto improve immunogenicity of tumor cell lysate vaccines.

TCLV are produced by mechanically or enzymatically gaining tumor cellsfrom tumor tissue originating from primary tumor material, metastasesand the like to small pieces and by subsequently lysing and killing themby repeated freezing and thawing—in contrast to whole cell vaccines(WCV). The lysates produced in that manner are so-called “necroticlysates” (Gallucci (1999), Nat. Med., 11, 1249-55; Kotera (2001), loc.cit.; Restifo (2000), loc. cit.; Sauter (2000), J. Ex. Med., 191,423-34).

During the process of the “real” necrotic death a series of genes areactivated which are only fragmentally known, however, resulting in thesecretion of proteins into the medium which show a pro-inflammatoryresponse. However, altered activation of gene expression is highlyunlikely to take place during the rapid freeze/thawing as described inthe prior art. Therefore, the above-mentioned positive effects assumedto be connected with “real” necrotic cells (Melcher (1999) J. Mol. Med77, 824-833) and do not apply to the allegedly “necrotic” cell lysates.Thus, the allegedly “necrotic” cell lysates of the prior art do in factnot comprise relevant necrotic cells in the sense of the invention.

Heat shock proteins are amongst the gene products, Which are suggestedto be involved in the immunological effects of necrotic cells on theimmune system (Gallucci (2001), Curr. Opin. Immunol. 13, 114-119; Todryk(2000), loc. cit.). By now many signals have been identified which causean increased expression of HSPs. Heat shock proteins (HSPs), belong tothe group of stress proteins which are present in all cells in all lifeforms. They are induced when a cell undergoes various types ofenvironmental stresses like heat, cold and oxygen deprivation.

HSPs are also present in cells under perfectly normal conditions. Theyact like “chaperones,” making sure that the cell's proteins are in theright shape and in the right place at the right time. For example, HSPshelp new or distorted proteins fold into shape, which is essential fortheir function. They also shuttle proteins from one compartment toanother inside the cell, and transport old proteins to “garbagedisposals” inside the cell.

For decades it has been known that animals, e.g. mice can be“vaccinated” against cancer and after many experiments, it was foundthat one element responsible for immunological responses in mice wereheat shock proteins. In particular Hsp70 expression has been shown to belinked with induction of cell death. Thus, Hsp70 seems to be at leastone important factor involved in the immunstimulatory effects ofnecrotic cells (Dressel (2000), J. Immunol. 164, 2362-2371; Melcher(1998), loc. cit.; Todryk (2000), loc. cit.). Feng (Feng (2001), Blood11, 3505-3512) has induced the expression of membrane-bound Hsp70 incells, which caused improvement of the immunogenicity in an animal modelsystem. In a control experiment these cells have been injected as alysate into mice without provoking an anti-tumor response. Furthermore,the immunstimulatory role of certain isolated HSPs, in particular Hsp70and gp96, has been demonstrated (U.S. Pat. No. 6,168,793; U.S. Pat. No.5,948,646; U.S. Pat. No. 5,961,979; Schild (2000), Nat. Immunol. 1,100-101; Binder (2000), J. Immunol. 165, 6029-6035; Wells (2000),Immunol. Today 21, 129-132). In addition, HSPs purified from celllysates have also been described as highly immunogenic molecules, whichpresent peptides to the immune system. Therefore, they are usedcommercially for the development of tumor vaccines.

In conclusion, there are problems or drawbacks associated with wholecell vaccines (WCV) or hyperthermia and with tumor cell lysate vaccines(TCLV) and in order to develop or to obtain potent vaccines it isimportant to increase the immunogenicity dramatically. The medical needand commercial interest for an according efficient tumor vaccine, whichcan be produced easily, cost efficiently and in a highly reproducibleway is therefore given.

Thus, the technical problem underlying the present invention is toprovide means and methods for improved vaccination against cancers,tumorous diseases, infections and/or autoimmune diseases.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a process for theproduction of an immunogenic compound comprising the steps of

(a) inducing necrosis by temperature in tumor cells; and

(b) lysing said necrotic tumor cells so as to obtain a lysate.

Surprisingly it was found that induction of necrosis by temperatureleads to an increased immunogenicity of tumor cell lysates both in vitroand in vivo. Even more surprising was the finding that tumor celllysates harbouring a large amount of heat shock proteins are not asimmunogenic as tumor cell lysates which were generated with theinventive process harbouring an amount of heat shock proteins comparableto that of tumor cell lysates from untreated, i.e. non-necrotictumor-cells. This indicates that the expression of heat shock proteinsis not correlated with the increased immunogenic effect of the lysatesaccording to the invention.

According to the present invention the term “tumor cells” means celllines or cells which can be grown under in vitro culture conditions, ortumor cell lines and primary cell cultures, or tumor cells derived fromprimary or secondary tumor or metastases as listed herein. The cells canbe of autologous, allogeneic, syngenic, or xenogenic origin in relationto the person, patient or animal treated and from the same or fromdifferent tissues, organs or cell origin in a species (e.g. in case ofcancer treatment or prevention in relation to the treated or to beprevented cancer type). The tumor cells used in the process can also bemixtures of the above-mentioned tumor cells. In a preferred embodimentthose tumor cells can be altered via mutagenesis, infection withpathogenic particles, like viruses, bacteria, fungi, parasites, or viagentechnological methods, thereby introducing novel antigens orimmunogens or parts thereof. In particular, the introduced antigens orimmunogens or parts thereof can be of tumors or infectious diseasesorigin or can be connected with these diseases.

In accordance with the present invention, the term “necrotic tumorcells” means a cell population containing at least 15% necrotic cells asdetermined for example by the techniques mentioned hereinbelow.

When the tumor cells are derived from tumors or metastases, alsoincluding micrometastases, they can, e.g., be obtained by surgery,biopsy, or the like.

The tumor cells can be derived from any possible type of tumors.Examples are skin, breast, brain, cervical carcinomas, testicularcarcinomas, head and neck, lung, mediastinum, gastrointestinal tract,genitourinary system, gynaecological system, breast, endocrine system,skin, childhood, unknown primary site or metastatic cancer, a sarcoma ofthe soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of thecentral nervous system, a lymphoma, a leukaemia, a paraneoplasticsyndrome, a peritoneal carcinomastosis, a immunosuppression-relatedmalignancy and/or metastatic cancer etc. The tumor cells may, e.g., bederived from: head and neck, comprising tumors of the nasal cavity,paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx,hypopharynx, salivary glands and paragangliomas, a cancer of the lung,comprising non-small cell lung cancer, small cell lung cancer, a cancerof the mediastinum, a cancer of the gastrointestinal tract, comprisingcancer of the oesophagus, stomach, pancreas, liver, biliary tree, smallintestine, colon, rectum and anal region, a cancer of the genitourinarysystem, comprising cancer of the kidney, urethra, bladder, prostate,urethra, penis and testis, a gynaecologic cancer, comprising cancer ofthe cervix, vagina, vulva, uterine body, gestational trophoblasticdiseases, ovarian, fallopian tube, peritoneal, a cancer of the breast, acancer of the endocrine system, comprising a tumor of the thyroid,parathyroid, adrenal cortex, pancreatic endocrine tumors, carcinoidtumor and carcinoid syndrome, multiple endocrine neoplasias, a sarcomaof the soft tissue and bone, a mesothelioma, a cancer of the skin, amelanoma, comprising cutaneous melanomas and intraocular melanomas, aneoplasm of the central nervous system, a cancer of the childhood,comprising retinoblastoma, Wilm's tumor, neurofibromatoses,neuroblastoma, Ewing's sarcoma family of tumors, rhabdomyosarcoma, alymphoma, comprising non-Hodgkin's lymphomas, cutaneous T-celllymphomas, primary central nervous system lymphoma, and Hodgkin'sdisease, a leukaemia, comprising acute leukemias, chronic myelogenousand lymphocytic leukemias, plasma cell neoplasms and myelodysplasticsyndromes, a paraneoplastic syndrome, a cancer of unknown primary site,a peritoneal carcinomastosis, a immunosuppression-related malignancy,comprising AIDS-related malignancies, comprising Kaposi's sarcoma,AIDS-associated lymphomas, AIDS-associated primary central nervoussystem lymphoma, AIDS-associated Hodgkin's disease and AIDS-associatedanogenital cancers, and transplantation-related malignancies, ametastatic cancer to the liver, metastatic cancer to the bone, malignantpleural and pericardial effusions and malignant ascites. It is mostlypreferred that said cancer or tumorous disease is cancer of the head andneck, lung, mediastinum, gastrointestinal tract, genitourinary system,gynaecological system, breast, endocrine system, skin, childhood,unknown primary site or metastatic cancer, a sarcoma of the soft tissueand bone, a mesothelioma, a melanoma, a neoplasm of the central nervoussystem, a lymphoma, a leukemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer. Accordingly, the term “tumor cell” as providedherein, includes in particular a cell afflicted by any one of theabove-identified conditions, but is not limited to the mentionedconditions.

The tumor cells are provided in step (a) of the process according to theinvention in a form which allows to induce necrosis by temperature.

The tumor cells may be in any state or form, which allows inducingnecrosis. They may, e.g., be in the form of fresh material or can betaken from previously frozen material. In a preferred embodiment thepreviously frozen material in form of cell lines is thoroughly thawedand cultivated. If tumor material is used, it is preferably materialwhich has already been treated mechanically and/or enzymatically so asto provide smaller tissue pieces and/or separate cells.

The resulting cells or tissue pieces are treated afterwards bytemperature in order to induce necrosis or are cultivated for a suitabletime in vitro.

In the context of the present invention the term “necrosis” meansmorphological changes of cells. Necrosis is, inter alia, characterizedfor example by “leakiness” of the cell membrane, i.e. an increasedpermeability which also leads to an efflux of the cell's contents and aninflux of substances perturbing homeostasis and ion-equilibrium of thecell, DNA fragmentation and, finally, to the generation of granularstructures originating from collapsed cells, i.e. cellular debris.Typically, necrosis results in the secretion of proteins into thesurrounding which, when occurring in vivo, leads to a pro-inflammatoryresponse.

Methods for the determination whether a cell is necrotic or not areknown in the prior art and are also described in the examples herein. Itis not important which method the person skilled in the art choosessince various methods are known.

However, it is important to distinguish between an apoptotic cellundergoing the so-called programmed cell death and a necrotic cell.Necrotic cells in accordance with the present invention can bedetermined, e.g., by light-, fluorescence or electron microscopytechniques, using, e.g., the classical staining with trypan blue,whereby the necrotic cells take up the dye and, thus, are stained blue,or distinguish necrotic cells via morphological changes including lossof membrane integrity, disintegration of organelles and/or flocculationof chromatin. Other methods include flow cytometry, e.g., by stainingnecrotic cells with propidium iodide. The preferred propidium iodidestaining is described in detail in the examples herein. Apoptotic cellscan be determined, e.g., via flow-cytometric methods, e.g., attainingwith Annexin V-FITC, with the fluourchrome: Flura-red, Quin-2, with7-amino-actinomycin D (7-AAD), decrease of the accumulation of Rhodamine123, detection of DNA-fragmentation by endonucleases: TUNEL-method(terminal deoxynucleotidyl transferase caused X-UTP nick labelling), vialight microscopy by staining with Hoechst 33258 dye, via Western blotanalysis, e.g., by detecting caspase 3 activity by labelling the 89 kDaproduct with a specific antibody or by detecting the efflux ofcytochrome C by labelling with a specific antibody, or via agarose gelDNA-analysis detecting the characteristic DNA-fragmentation by aspecific DNA-ladder.

The preferred technologies are described in detail in the examplesherein, and are the propidium iodide staining for necrotic cells and theannexin staining for apoptotic cells, whereby apoptotic cells are onlystained by annexin V and necrotic cells are stained by propidium iodideand annexin V (Vermes (1995), J. Immun. Meth. 184, 39-51). Thisflow-cytometry allows rapid and easy qualitative and quantitativemeasurements.

Preferably, necrosis is determined by flow cytometry, even morepreferred is flow cytometry using Annexin V/propidium iodid staining andmost preferred is the method as described in the examples herein.

The term “inducing necrosis by temperature” in the context of thepresent invention means that in a percentage of at least 15%; preferablyin more than 40% and herein preferred at least in 70% of the cells ofthe cell population of the tumor cells changes which result in necrosis.

Methods for measuring said temperature, which is used for inducingnecrosis, are known to the person skilled in the art. Preferably, saidtemperature can be measured by physical means. Said physical means ispreferably an optical, mechanical or electrical thermometer, whereby itis understood that normal physical fluctuation in the measurement of atemperature due to errors in the measurement or physical inertia of themeans are in the acceptable range of measurement tolerances. Mostpreferably, said physical means for measuring temperatures areincorporated by the supplier in the means or in the apparatus used forheat-induction. However, it is also preferred that the temperature maybe measured directly within the sample of tumor cells undergoingtemperature-induction.

The temperature for inducing necrosis in the cells can be applied to thecells by means and methods known to the person skilled in the art.Possible methods are, e.g., the incubation in heated air or water orirradiation. Suitable means are, e.g., heating blocks, thermal heaters,water bathes incubators, heating rods and the like.

It was surprisingly found that cell lysates produced from tumor cells inwhich necrosis was induced by temperature as described herein aresuperior to those of the prior art since their immunogenicity isincreased significantly.

It is important to note that the necrotic cell lysates consist of cellpopulations with the mentioned percentages of relevant necrotic cells asdefined above, while cell lysates, which are sometimes called necroticcell lysates in the literature and which correspond to cells lyseddirectly without induction of necrosis via rapid freeze/thawing, arecalled non-treated cells or non-treated cell lysates in the examplesherein.

In accordance with the present invention the term “lysing” relates tovarious methods known in the art for opening/destroying cells. Themethod for lysing a cell is not important and any method that canachieve lysis of the tumor cells may be employed. An appropriate one canbe chosen by the person skilled in the art, e.g. opening/destruction ofcells can be done enzymatically, chemically or physically. Non-limitingexamples for enzymes and enzyme cocktails are proteases, like proteinaseK, lipases or glycosidases; non-limiting examples for chemicals areionophores, like nigromycin, detergents, like sodium dodecyl sulfate,acids or bases; and non-limiting examples of physical means are highpressure, like French-pressing, osmolarity, temperature, like heat orcold. Additionally, a method employing an appropriate combination of anenzyme other than the proteolytic enzyme, an acid, a base and the likemay also be utilized.

According to the present invention the term “lysate” means a solution orsuspension in an aqueous medium of cells that are broken. However, theterm should not be construed in any limiting way. The cell lysatecomprises, e.g., macromolecules, like DNA, RNA, proteins, peptides,carbohydrates, lipids and the like and/or micromolecules, like aminoacids, sugars, lipid acids and the like, or fractions of it.Additionally, said lysate comprises cell debris which may be of smoothor granular structure. The details of the preparations are specificallydescribed in the examples of the present specification. Accordingly,those skilled in the art can prepare the desired lysates by referring tothe above general explanations and specific explanations in theexamples, and appropriately modifying or altering those methods, ifnecessary. Preferably, said aqueous medium is water, physiologicalsaline, or a buffer solution that any solid mass cannot be observedwithout help of optical means, and that the dispersoids can bephagocytosed by the antigen-presenting cells. An advantage of the tumorcell lysate obtainable by the processes of the present invention is thatit can be easily produced as described in the appended Examples andstored cost efficiently since less technical facilities are needed.

However, said lysate is not limited to necrotic cells since, forexample, due to the different sensitivity of the treated cells or due tothe applied conditions for the temperature-induction of cells alsoapoptotic cells can be part of the cell population from which the lysateis obtained. Nevertheless, necrotic cells have to be at least 15% of thecell population, preferably more than 40%, particularly more than 70%.The determination of the percentages may vary from experiment toexperiment due to measuring inaccuracies occurring in the technology offlow cytometry and the possibility of varying interpretations of thedata and setting of the gates. Accordingly, a deviation of 10%,preferably of 5% and more, preferably of 1% may occur if a method otherthan that specifically described in the examples is used.

Preferably, the tumor cells are lysed by freezing and thawing, morepreferably freezing at temperatures below −70° C. and thawing attemperatures of more than 30° C., particularly freezing is preferred attemperatures below −75° C. and thawing is preferred at temperatures ofmore than 35° C. and most preferred are temperatures for freezing below−80° C. and temperatures for thawing of more than 37° C. It is alsopreferred that said freezing/thawing is repeated for at least 1 time,more preferably for at least 2 times, even more preferred for at least 3times, particularly preferred for at least 4 times and most preferredfor at least 5 times.

According to the invention, lysates are also preparations of fractionsof molecules from the above-mentioned lysates. These fractions can beobtained by methods known to those skilled in the art, e.g.,chromatography, including, e.g., affinity chromatography, ion-exchangechromatography, size-exclusion chromatography, reversedphase-chromatography, and chromatography with other chromatographicmaterial in column or batch methods, other fractionation methods, e.g.,filtration methods, e.g., ultrafiltration, dialysis, dialysis andconcentration with size-exclusion in centrifugation, centrifugation indensity-gradients or step matrices, precipitation, e.g., affinityprecipitations, salting-in or salting-out(ammoniumsulfate-precipitation), alcoholic precipitations or otherproteinchemical, molecular biological, biochemical, immunological,chemical or physical methods to separate above components of thelysates. In a preferred embodiment those fractions which are moreimmunogenic than others are preferred. Those skilled in the art are ableto choose a suitable method and determine its immunogenic potential byreferring to the above general explanations and specific explanations inthe examples herein, and appropriately modifying or altering thosemethods, if necessary.

The induction of necrosis by temperature according to step (a) of themethod according to the invention is preferably achieved by incubationof the tumor cells at a temperature which is above the average bodytemperature of the organism from which the cells are derived, preferablyat a temperature above 37° C. More preferably, the cells are incubatedat a temperature of more than 38° C., even more preferably of more than39° C., particularly preferred of more than 40° C. and most preferred ofmore than 41,5° C.

It is preferred that the temperature is chosen in a way that althoughnecrosis is induced, no or only a low increase in hsp70 expression isinduced. By a “low increase” an increase of preferably not more than afactor of 2 is meant. The corresponding temperature can depend on thespecific cell type used but can be easily determined by the skilledperson by routine experimentation, e.g. by incubating the correspondingcells at different temperatures and determining the amount of necroticcells and the level of hsp70 expression at different temperatures usingmethods as those shown in the examples herein.

In an even more preferred embodiment the tumor cells are incubated at atemperature of more than 41.2° C., more preferably at a temperature ofmore than 42° C., more preferably at a temperature in the range of 45°C. to 55° C., even more preferred in the range of 45.5° C. to 47° C. Ina most preferred embodiment the cells are incubated at a temperature inthe range of 46.0° C. to 46.4° C., in particular at about 46.2° C.

As shown in the examples herein, temperatures of about 46.2° C. used forinducing necrosis lead to a surprisingly high amount of necrotic cellsand an unexpectedly high increase in immunogenicity.

Moreover, it is demonstrated in the examples herein that tumor celllysates produced from tumor cells in which necrosis has been induced bytemperature of about 46.2° C. have also surprising effects in vivo.Comparable results were also achieved with cell lysates from the samecell line induced at 46° C.

In particular, mice immunized with the tumor cell lysates of the presentinvention showed immune reactions against the antigens present on thetumor cells used for the production of the cell lysates. Said micedeveloped antibodies of the IgG-type that can only be produced if aso-called class-switch has occurred. Said class-switch is only possibleif T helper cells are included in the immune response to antigens.Moreover, it was also surprisingly found that the immunized micedeveloped said IgG-type antibodies against carbohydrate antigens forwhich so far only IgM-type antibodies have been developed by mice.However, IgM-type antibodies can be produced without a class-switch thatimplies that T helper cells had not been involved. Thus, the tumor celllysates of the present invention are advantageous as regardsimmunogenicity and are favourable for vaccination against cancer,tumorous diseases, infections and/or autoimmune diseases since theyevoke an immune response in which also B- and T-cells are involved.

The incubation of the tumor cells at a specific temperature is carriedout for a period of time sufficient to induce necrosis and depends onthe temperature and the means and methods for producing it. It is ofnote that the person skilled in the art knows that the time needed forinduction of necrosis in said tumor cells may vary depending, interalia, on the cells used, the status of the cells, the conditions in theculture medium, the sensitivity of the cells and the like. Moreover, thetime for induction of necrosis may also depend on the kind of applyingthe temperature to the cells and on the apparatus used for applying thetemperature. Moreover, the time for inducing said necrosis is alsodependent on the temperature at which the cells have been incubated forthe induction of necrosis. The most preferable temperature may also varydepending on the type and source of the tumor cells.

Generally, the incubation should last for a period of time of at least 1minute. Preferably, the induction lasts for a period of time of at leastn minutes, wherein n is an integer in the range of 2 to 60, with n=15being particularly preferred. More preferably, the incubation lasts atleast 1 hour, even more preferred at least 2 hours and particularlypreferred at least 3 hours. More preferably between 1.5 and 5 hours. Themost preferred incubation is between 2 and 3 hours.

There is in principle no upper limit for the time of incubation.However, it is preferably no longer than 48, 36, 24, 12, 11, 10, 9, 8,7, 6, 5 or 4 hours.

In an advantageous preferred embodiment the necrotic cells are directlylysed after the temperature induction of necrosis aftertemperature-induction of necrosis.

In a preferred embodiment the necrotic cells obtained after step (a) ofthe process according to the invention are further incubated beforelysing them. As shown in the examples herein, this further incubationalso influences the amount and immunogenicity of necrotic cells. Saidfurther incubation is preferred to be performed at a temperature of morethan 35° C., even more preferably at a temperature of more than 36° C.,particularly preferred at a temperature of about 37° C. The time forthis further incubation is preferred to be at least 0.1 hour, morepreferably to be at least 2 hours, even more preferred to be at least 3hours, particularly preferred to be at least 6 hours, even moreparticularly preferred to be at least 12 hours and most preferred to beat least 22 hours.

In a preferred embodiment of the invention, it is envisaged that in theprocess of the present invention more than 15% of said induced tumorcells are necrotic.

As mentioned herein above, due to, e.g., the different sensitivity ofthe treated cells, due to the applied conditions for thetemperature-induction of cells or due to different conditions inculturing the tumor cells that consist of a population of the cells alsocomprising apoptotic cells which are subsequently lysed together withthe other cells of the population also comprising the necrotic cellsaccording to the described process.

In order to obtain an effective immunogenicity it is desirable to obtaina certain percentage of necrotic cells in the lysate. Accordingly, theprocess is preferably designed so as to result in more than 15, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86,87, 88, 89, 90, 92, 94, 96, 98, 99% of the temperature-induced tumorcells being necrotic. Most preferably, more than 40% or 70% of saidtumor cells are necrotic. It is of note that the amount of necroticcells can also depend on the temperature, time and time for regenerationafter induction of necrosis by temperature as demonstrated in theexamples herein as well as on the type and source of the tumor cells.

Also preferred is that the tumor cells used in the process according tothe invention are genetically engineered, mutated or infected byoncogenic viruses.

In the context of the present invention the term “geneticallyengineered” is used in its broadest sense for methods known to theperson skilled in the art to modify desired nucleic acids in vitro andin vivo such that genetic modifications are affected and genes arealtered by recombinant DNA technology. Accordingly, it is preferred thatsaid methods comprise cloning, sequencing and transformation ofrecombinant nucleic acids. For this purpose appropriate vectors,primers, enzymes, host cells and the like can be used and are known bythe skilled artisan. Preferably, genetically engineered tumor cellscomprise cells harbouring recombinant nucleic acids encoding antigens orimmunogens or parts thereof, cytokines, chemokines, growth factors andthe like. Antigens and immunogens can be, for example, one or more tumorantigens or parts thereof, antigens from infectious microorganisms orparasites, like bacteria, fungi, viruses and the like. Furthermore,among the immunogens are, for example, molecules which increase theimmunogenicity, like pan T-cell epitopes or multimers thereof, likePADRE-epitopes, or tetanus toxoid fragments which evoke an additionalimmunstimulatory effect via activation of MHC class II-mediatedprocesses.

Also preferred are tumor cells genetically engineered with nucleic acidsencoding effector molecules, like transcription factors, components ofsignal transduction pathways or signalling cascades, or cytokines,chemokines, growth factors and the like which are able to modulatedirectly or indirectly the expression of endogenous molecules, e.g.nucleic acids, polypeptides, posttranslationally modified polypeptidesand lipids and the like. More preferably, the tumor cells aretransiently or stably transfected with a desired nucleic acid molecule.

It is also preferred that the tumor cells are genetically engineered soas to express a polypeptide against which antibodies should be raised.If cell lysates from these tumor cells are produced and administered toan individual, it is expected that a humoral and/or cellular immuneresponse is developed by individuals, preferably this immune responsecomprises antibody responses and/or T helper cell responses and/orcytotoxic T cell responses.

In accordance with the present invention, the term “mutated” means (a)permanent modification(s) of genetic material, i.e. nucleic acids,caused, for example, naturally or by physical means or chemicalcompounds/substances/agents. Said modifications include point mutations,like transitions or transversions, deletion/insertion/addition of one ormore bases within a nucleic acid/gene/chromosome thereby modifying thenucleic acid/gene/chromosome which can cause, inter alia, aberrant geneexpression/transcription/translation or inactive gene products,constitutive active/inactive gene products leading to e.g.dominant-negative effects. Thus, it is also preferred that the tumorcells comprise cells which harbour (a) mutation(s) in (a) desiredgene(s) or in which (a) mutation(s) in (a) desired gene(s) is induced bymethods known to the person skilled in the art. It is also known in theprior art that mutated or genetically engineered tumor cells can beselected by any suitable method/phenotype.

In accordance with the present invention the term “infected” meanscells, which have been infected with a virus, or viroid, and/orproteinaceous structure. Said virus, or viroid, and/or proteinaceousstructure may also be used as a vehicle for genetically engineering saidcells. It is preferred that said virus which infects tumor cells is anoncogenic virus, however, is not limited to oncogenic viruses. Mostpreferably said oncogenic virus is selected from the group consisting ofretroviruses or DNA viruses, e.g. papovaviruses like human papillomaviruses (HPV), type C oncoviruses, like human T cell leukaemia viruses(HTLV), herpes viruses, like Epstein-Barr virus (EBV), hepadnaviruses,like hepatitis B virus (HBV), and lentiviruses, like human deficiencyvirus (HIV). It is also preferred that tumor cells are already infectedwith any one of the above mentioned viruses. Furthermore, infected tumorcells or lysates thereof may be important when used forprophylactic/therapeutic vaccination against infectious diseases causedfor example by viruses like HIV, HBV, hepatitis C virus (HCV), HPV.Preferably, the infectious component(s) comprised by the lysatesproduced from these infected cells has/have to be additionallyinactivated. Methods to be used are known to those skilled in the art,e.g., heat inactivation, acid inactivation and/or sterile filtration orthe like.

Moreover, in another preferred embodiment of the present invention thetumor cells are autologous.

In the context of the present invention the term “autologous” means thatthe tumor cells are derived from the same individual to which the lysateresulting from the process according to the invention shall be lateradministered. One advantage is by using cells from the autologous tumorthat there are a large variety of relevant and suitable antigens in thelysate for treating cancers or tumorous diseases of the individual orprevent its recurrence or relapse.

Alternatively, in another preferred embodiment the tumor cells areallogeneic. In the context of the present invention the term“allogeneic” means that the tumor cells are derived from an individualwhich is different from the individual to which the lysate resultingfrom the process according to the present invention shall be lateradministered. A herein further preferred embodiment of the allogeneictumor cells for use in an allogeneic setting are tumor cells of apatient, or cells originating from these cells, which were successfullyused for immunization of that latter patient in a autologous settingresulting in a partial or complete reduction of tumor load.

In the context of the present invention the term “allogeneic tumorcells” further comprises cell lines, including cell lines, e.g., tumorcell lines, or cell lines or cultures from primary material and thelike, which are not originating from the individual to which the lysateshall be administered. The advantage of allogeneic tumor cells areantigens or immunogens which are not shared by the cancers or tumorousdiseases to be treated or prevented, resulting in an immune responsecomprising a strong danger signal and/or helper response which can befavourable to overcome anergies or tolerances.

Alternatively, in a preferred embodiment the tumor cells are xenogenic.In the context of the present invention the term “xenogenic” means thatthe tumor cells comprise tumor cells from primary or secondary tumors orfrom metastases, cell lines, e.g., tumor cell lines, immortalized celllines or cell lines or cultures from primary material and the like,which are not originating from the same species to which the lysateshall be later administered.

Alternatively, in another preferred embodiment the tumor cells areallogeneic, autologous or xenogenic and are of a different tissue orcell source or the like than the cancers or tumors, e.g. tumor cellsfrom a mammary tumor cell line are used for generating a lysateaccording to the process of the invention which is administered to anindividual for prophylaxis or treatment of colon or gastric cancers ortumorous diseases, or a lysate according to the invention from a mutatedmyeloma cell line for use in an individual for prophylaxis or treatmentof carcinomas. Preferably, these cell lines have one or more antigenswhich are shared with the cancers or tumorous diseases to be treated.The advantage is an additional strong response against antigens orimmunogens foreign to the cancers or tumorous diseases to be treated orprevented, e.g., antigens specific for the tumor cell which are notshared by the cancer or tumors to be treated or prevented, comprising astrong danger signal and/or helper response which can be favourable toovercome anergies and/or tolerances. In a preferred embodiment the tumorcells are in addition from an allogeneic source which can in additionhave a strong allo-response which can be further favourable to overcomeanergies and/or tolerances.

In a preferred embodiment, the lysate is prepared from tumor cells ofdifferent types or different lysates prepared from different types oftumor cells are used in combination for the administration to anindividual.

More preferably the tumor cells of the lysate or the combination oflysates are allogeneic and autologous tumor cells. Even more preferably,they are a mixture from tumor cells from the same tissue or cell sourceand the like together with those from a different tissue or cell sourceand the like. It is, e.g., possible to use a cell lysate from anallogeneic mutated myeloma cell line with a lysate from colon cancercells from the individual to which the lysate(s) shall be lateradministered. The advantage is the combination of large amounts ofshared antigens from autologous material with the allogeneic part and,therefore, an increased helper, response, allo-response and/or dangersignals in order to break potential tolerances and/or anergies of thecancers or tumors. Another example is the mixture of the aboveallogeneic myeloma cell line with another colon or gastric cancer cellline for the administration to an individual to treat or prevent coloncarcinoma. The advantage of the latter is the possibility to generate apotent “off-the-shelf” vaccine with a mixture of large amounts of tumorantigens and an increased helper response, allo-response and/or dangersignals in order to break potential tolerances and/or anergies.

The tumor cells can be mixed before the induction of necrosis bytemperature induction or after induction but before the lysis or afterthe lysis. The skilled artisan is able to determine which is favourablefor the use for the production, regulatory issues, application to anindividual and for its cancers or tumorous diseases.

The examples of the present invention demonstrate that both, anautologous and allogeneic system, can be used for increasing theimunogenicity. As described hereinabove, the process of the presentinvention allows to produce highly efficient immunogenic compounds byinducing necrosis in the tumor cells and subsequently lysing the cells.The obtained tumor cell lysates can be used for the therapeutic orprophylactic treatment of cancer, tumorous diseases, infections and/orautoimmune diseases.

In this contex the term “immunogenic compound” means compounds havingthe ability to evoke immune reactions of the cells of the immune systemlike macrophages, dendritic cells, Langerhans' cells; B-(B1 and B2)and/or T-cells (cytotoxic T cells (Tc), T-helper cells (Th0, Th1 andTh2)), natural killer cells (NK cells), memory cells and the likePreferably, the term “immunogenic compound” means compounds having theability to evoke humoral and/or cellular immune response, wherein atleast one of the cells/group of cells of immune effector cells or cellproducts of said immune effector cells, for example one or more of theaforementioned cells are involved. The person skilled in the art isaware of various methods to determine whether an immune response isevoked. Examples for methods used for this purpose are shown in theexamples and be transferred, where necessary by those skilled in theart, to determine the response in an individual or patient treated withthe lysates according to the invention. Furthermore, other methods knownto those skilled in the art can complement these techniques. In general,an immunogenic compound leads to an immune response comprising humoraland/or cellular responses, normally comprising that genes or geneproducts that affect the level of immune responses areexpressed/activated, e.g. those of the major histocompatibility class(MHC) I and II, those of antibody light and heavy chains, those ofmembers of the immunglobulin superfamily, those of T-cellreceptor/receptor compounds, those of cytokines or those of signaltransduction cascades involved in transmitting immune responses.

In a preferred embodiment, the tumor cells used for the production of acell lysate as described herein are NM-F9 (DSMZ deposit No. DSM ACC2606or NM-D4 cells (DSMZ deposit No. DSM ACC2605)

The term “NM-F9” (also referred herein as “F9” or “TF-positive F9cells”) or “NM-D4” means cell lines or cells derived from the humanmyelogenous leukemia cell line K562. (ATCC: CCL-243). NM-F9 and NM-D4were deposited with the Deutsche: Sammlung für Mikroorganismen undZellkulturen GmbH (“DSMZ”) on Aug. 14, 2003. The DSMZ is located at theMascheroder Weg 1b, D-38124 Braunschweig, Germany. The DSMZ deposit wasmade pursuant to the terms of the Budapest treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

The present invention also relates to a lysate obtainable or obtained bythe process according to the present invention and to dendritic cellsloaded with such a lysate.

Moreover, the present invention also relates to a composition comprisinga lysate or dendritic cells according to the present invention.

In a preferred embodiment said composition is a pharmaceuticalcomposition. In accordance with the present invention the term“pharmaceutical composition” relates to compositions comprising the celllysates described hereinabove which are obtained by the aforementionedprocesses and having the desired pharmacological activity. Suchpharmaceutical compositions comprise a therapeutically effective amountof the cell lysates of the present invention, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition may be administeredwith a physiologically acceptable carrier to a patient, as describedherein. In a specific embodiment, the term “pharmaceutically acceptable”means approved by a regulatory agency or other generally recognizedpharmacopoeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil; sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecell lysate, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilised powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration. The cell lysate of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

The amount of the cell lysate of the invention which will be effectivein the treatment or prevention (in particular by vaccination) ofcancers, tumors, tumorous diseases, infections and/or autoimmunediseases can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

In another preferred embodiment the composition is a vaccinecomposition.

In accordance with the present invention the term “vaccine composition”relates to any composition which can be used as a vaccine.

The forms or methods for manufacturing vaccine compositions according tothe present invention are not particularly limited, and a composition ina desired form can be prepared by applying a single method available inthe field of the art or methods in an appropriate combination. For themanufacture of a vaccine composition, aqueous media such as distilledwater for injection and physiological saline, as well as one or morekinds of pharmaceutical additives available in the field of the art canbe used. For example, buffering agents, pH adjusting agents,solubilizing aids, stabilizing agents, soothing agents, antiseptics andthe like can be used, and specific ingredients thereof are well known tothose skilled in the art. The vaccine composition can also be preparedas a solid preparation such as a lyophilized preparation, and thenprepared as an injection by adding a solubilizing agent such asdistilled water for injection before use. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways asdiscussed below. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100 wt %. The vaccinecomposition may be administered alone or in combination with othertreatments, i.e., radiation, or other chemotherapeutic agents oranti-cancer agents.

In a preferred embodiment, the vaccine compositions are in awater-soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The vaccine compositions can be prepared in various forms, such asinjection solutions, tablets, pills, suppositories, capsules,suspensions, and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The vaccine compositions may also include one or more of the following:carrier proteins such as serum albumin; buffers; fillers such asmicrocrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavouring agents; colouring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

A vaccine composition according to the present invention can be used forimmunization against cancer, tumorous diseases, autoimmune diseasesand/or infectious diseases.

In another preferred embodiment the composition according to the presentinvention, in particular the vaccine composition, is optionally combinedwith an adjuvant or dendritic cells.

In accordance with the present invention “dendritic cells” relate toprofessional antigen-presenting cells which capture antigens and migrateto the lymph nodes and spleen, where they are particularly active inpresenting the processed antigen to T cells. The term “dendritic cells”also means cells which have an activity and function similar todendritic cells. Dendritic cells can be derived from either the lymphoidor mononuclear phagocyte lineages. Said dendritic cells can be found inlymphatic and non-lymphatic tissue. The latter appear to induce a T cellresponse only when being activated and having migrated to lymphatictissues.

Dendritic cells are known to be the or amongst the most potentactivators and regulators of immune responses. One important feature isthat they are presently the only antigen presenting cells known tostimulate naive T cells. Immature dendritic cells are characterized bytheir ability to take-up and process antigens, a function that isdramatically reduced in mature dendritic cells, which in turn exhibitenhanced presentation of processed antigens on their surface, mainlybound to MHC Class I and Class II molecules. Maturation is alsoassociated with upregulation of costimulatory molecules (such as CD40,CD80 and CD86), as well as certain other cell surface proteins (e.g.CD83 and DC-Sign). Dendritic cell maturation is also usually associatedwith enhanced migratory capacity, resulting (in vivo) in migration ofdendritic cells to the regional lymph nodes, where the dendritic cellsencounter T and B lymphocytes. Dendritic cells can be obtained fromindividuals using methods known to those skilled in the art and aredescribed in more detail in the examples herein. Furthermore, accordingto the invention, dendritic cells are also those cells or cell lineswhich show the comparable functional and/or phenotypic features asdendritic cells, e.g. MUTZ-3 derived cells.

Dendritic cells or their precursors are differentiated using suitablegrowth factors and/or cytokines, e.g. GM-CSF and IL-4 as shown in theexamples herein, the resulting immature dendritic cells are loaded witha lysate according to the invention. Immature DC (iDC) loaded with alysate according to the invention are further maturated to mature DC(mDC). In special cases also mDC can be loaded (pulsed) with antigens orimmunogens from the lysate. Vaccine compositions or pharmaceuticalcompositions for preventing or treating cancers, tumorous diseases andor infectious diseases preferentially comprise loaded mDC whichoriginate from loaded and matured iDC or which were loaded after orduring maturation. Vaccine compositions for autoimmune diseasespreferentially comprise loaded iDC which are preferably transiently orstably arrested in the iDC state using methods known to those skilled inthe art.

In a preferred embodiment of the invention treatment or prevention ofthe cancers, tumorous diseases, infections and/or autoimmune diseasesand in particular for cancers, tumorous and infectious diseases combinesdosages comprising a lysate according to the invention loaded ontodendritic cells with dosages comprising only a lysate according to theinvention. The advantage is to combine the ex vivo loading of autologousor allogeneic dendritic cells with the in vivo “loading” of dendriticcells which occurs via the administration of the lysate to anindividual. Thereby, different application routes might be preferable.The administration of dendritic cells directly to lymphnodes or otherareas with direct contact to the important immune cells to be stimulatedby dendritic cells are preferred. The administration of the lysateintradermally, subcutaneously or intrarectally to Payers patches orother areas where dendritic cells or their precursors are located andpreferably concentrated, is preferred.

Another preferred embodiment of the invention are vaccine compositionsor pharmaceutical compositions comprising a lysate according to theinvention loaded onto suitable dendritic cells and adjuvants orcostimulatory factors for the enhancement of the action of the dendriticcells, e.g. GM-CSF, interleukins.

With respect to the present invention the term “adjuvant” means that thenatural ability of an antigen to induce an immune response can bemodified, and in particular enhanced, by altering or by mixing it orloaded dendritic cells described hereinabove with another substance. Theterm “adjuvant” also means that tumor cells from which the lysates aregenerated and/or dendritic cells are genetically modified in order toexpress adjuvants or other factors which influence the immune response,as for example costimulatory factors. The procedure or the substanceused- to enhance immune responses is called an adjuvant. At least threeclasses of adjuvants have been used for a long time; these are mineraloil emulsions, aluminium compounds, and surface active materials such assaponin, lysolecithin, retinal, Quil A.RTM., some liposomes, andpluronic polymer formulations. See, for example, Fundamental Immunology,edited by William E. Paul, at p. 1008, Raven Press, New York (this bookwill hereinafter be referred to as “Fundamental Immunology”). Aluminiumadjuvants used alone or in combination include aluminium hydroxide gel,aluminium phosphate, aluminium sulphate, and alums comprising ammoniumalum (such as (NH.sub.4).sub.2 SO.sub.4.Al.sub.2 (SO.sub.4).sub.3) andpotassium alum. Aluminium hydroxide (hereinafter “AL”) is one of theolder adjuvants and it is considered so safe that it has been applied inbacterial and viral vaccines administered to billions of people aroundthe world. Calcium phosphate gel (hereinafter “CP”) has similarproperties and is also used in vaccines. Both substances are availablein pharmaceutical qualities in most countries worldwide. Techniques forpreparing adjuvant-antigen preparations for injection are well known inthe art. See, for example, Terry M. Phillips, Analytical Techniques inImmunochemistry, pp. 307-10, Marcel Dekker, New York, 1992. Otheradjuvants include complete Freund's adjuvant (a water-in-oil emulsion inwhich killed, dried, mycobacteria—usually M tuberculosis—are suspendedin the oil phase); incomplete Freund's adjuvant (analogous to thecomplete Freund's adjuvant with no mycobacteria); ISCOM (or immunestimulating complex, comprising lipophilic particles formed by thespontaneous association of cholesterol, phospholipid and the saponinQuil A.RTM.); lipopolysaccharide (complex molecules consisting of alipid core—lipid A—with a polysaccharide side chain that are componentsof certain bacilli, Lipid A is incorporated into the outer membrane ofthe bacterium and the polysaccharide projects extracellularly. Theiradjuvant potency is associated with lipid A; they are also mitogenic formurine B lymphocytes); and mycobacterial adjuvants (whole, heat killed,dried, mycobacteria—such as M. tuberculosis, M. avium, M. phlei, and M.smegmatis) that, when suspended in mineral oil and emulsifier, haveadjuvant activity with respect to any antigen given with them. Extractsof some mycobacteria, e.g., mycobacterial peptidoglycolipids havesimilar adjuvant activities. See, for example, Dictionary of Immunologyat pp. 3, 7, 46, 94, 97, 105, and 116; R. B. Luftig, Microbiology andImmunology, pp. 228-29, Lippincott-Raven Publishers, Philadelphia 1998.Microbial adjuvants include Corynebacterium parvum and Bordetellapertussis. See, for example, Handbook of Immunology at 115-16. Use ofcontrolled-release preparations and materials with adjuvant activity andpossible sites of action have been described in Fundamental Immunologyat pp. 1007-09. Mineral carriers such as aluminium hydroxide, potassiumammonium sulphate, and potassium aluminium sulphate adsorb the antigenon their surface. These common adjuvants have been used at a 0.1%concentration with up to 1 mg protein antigen in 1 ml administered toanimals at doses of 0.2-0.5 ml/(kg body weight). See Miroslav Ferencik,Handbook of Immunochemistry, p. 115, Chapman & Hall 1993 (this book willhereinafter be referred to as “Handbook of Immunochemistry”). AlthoughFreund's adjuvant is toxic and not used for immunization of humanbeings, mineral adjuvants such as aluminium hydroxide are common inhuman medicine. Id. at 116. In addition to alum, other adjuvants in thegroup of inert carriers include bentonite, latex, and acrylic particles.See Fundamental Immunology at 1008. Combinations of adjuvants can alsohave adjuvant properties. For example, it has been shown that thecombination of saponin and muramyl dipeptide in a squalene in wateremulsion is superior to alum as an adjuvant for inducing certainresponses in mice. R. Bomford, M. Stapleton, S. Wilson, A. McKnight, andT. Andronova, The control of the antibody isotype responses torecombinant human immunodeficiency virus gp120 antigen by adjuvants,AIDS Res. Hum. Retroviruses Vol. 8(1992) pp. 1765 et seq. Theseadjuvants are complemented by new adjuvants that have been developedduring the last fifteen years. See, for example, Anthony C. Allison, TheRole of cytokines in the Action of Immunological Adjuvants, in VaccineDesign. The Role of cytokine Networks, edited by Gregory Gregoriadis andBrenda McCormack, NATO ASI Series A: Life Sciences Vol 293, pp. 1-9,Plenum Press, New York 1997 (this book will hereinafter be referred toas “Vaccine Design”); Immunology at p. 116; H. Snippe, I. M. Fernandezand C. A. Kraaijeveld, Adjuvant Directed Immune Specificity at theEpitope Level. Implications for Vaccine Development. A Model Study UsingSemliki Forest Virus Infection of Mice, in Vaccine Design at pp. 155-73.An adjuvant can be administered prior to, simultaneously with, orfollowing the administration of the antigen. Antibody productionenhancement caused by adjuvants is not fully understood. However,adjuvant properties that may exist either alone or in variouscombinations and which permit a substance or formulation to be describedas adjuvant active have been defined. See, for example, J. C. Cox and A.R. Coulter, Adjuvants—A classification and review of their modes ofaction, Vaccine Vol. 15(1981) pp. 248 et seq.; John Cox, Alan Coulter,Rod Macfarlan, Lorraine Beezum, John Bates, Tuen-Yee Wong and DebbieDrane, Development of an Influenza-ISCOM.TM. Vaccine, in Vaccine Designat pp. 3349. One of these properties is depot generation, whereby thevaccine is retained near the dose site to give short-term tricklerelease or a longer term pulsed release. Id. at p. 34.

Preferably, the pharmaceutical or vaccine composition is administereddirectly or in combination with an adjuvant mentioned herein above orloaded on antigen-presenting cells, particularly dendritic cells. It isalso preferred that both the pharmaceutical or vaccine composition andthe adjuvant and the pharmaceutical or vaccine composition and theloaded dendritic cells are administered together or separately from eachother e.g. at different time points or at different locations.Additionally, it is also preferred that said pharmaceutical compositionand adjuvant is administered together with said pharmaceuticalcomposition loaded on dendritic cells. Since dendritic cells are highlyspecialized antigen-presenting cells with the unique capability ininitiating and regulating antigen-specific immune responses, it ispreferred to combine them with the pharmaceutical or vaccinecompositions of the present invention. For the preparation of a tumorvaccine dendritic cells can be generated from the peripheral blood oftumor patients from other donors or from the above-mentioned cell lines.In clinical studies, the efficacy of vaccination with dendritic cellshas been demonstrated using immunological and—in some cases—clinicalendpoints.

Active specific immunotherapy approaches to the treatment of tumors havebeen widely investigated during recent years. Numerous studies involvingthe vaccination of patients with their own inactivated tumor cells havebeen reported. These studies have demonstrated that inclusion of anadjuvant is necessary to stimulate the patient's immune systemespecially against the autologous, or derived from self, tumor cells.For example, methods utilizing the particulate adjuvant, BacillusCalmette-Guerin (BCG) cells, administered systemically or mixed with thepatient's own tumor cells have been shown to induce tumor-specificimmunity in laboratory animals. Peters, L. C., Brandhorst, J. S., HannaJr., M. G., Preparation of Immuno-Therapeutic Autologous Tumor CellVaccines from Solid Tumors; Cancer Res. 39: 1353-1360 (1979).

In another preferred embodiment the dendritic cells used in theaforementioned pharmaceutical or vaccine composition are loaded maturedendritic cells (mDC) which originate from lysate-loaded and furthermatured immature dendritic cells (iDC) or which were loaded after orduring maturation. The term “immature” when used in accordance with thepresent application relates to professional antigen-presenting cellsthat are characterized by their ability to take-up and process antigens.The term “mature” when used in accordance with the present applicationrelates to professional antigen-presenting cells that expresscostimulatory factors and antigens in the context of MHC class moleculesor CD1 molecules and can activate T cells, regulatory NKT cells and/or Bcells.]

A pharmaceutical or vaccine composition comprising dendritic cells andtemperature-induced cell lysates, comprises them preferably as dendriticcells loaded with lysate. Said dendritic cells are preferably loaded intheir immature stadium (immature DC) with the cell lysates of thepresent invention and are subsequently being brought to maturation.Mature DC loaded with the lysate according to the invention arepreferably used to treat or prevent tumorous or infectious diseases. Incase, the pharmaceutical or vaccine composition is used for vaccinationagainst autoimmune diseases the immature DC are loaded and preferablyarrested in their immature stadium to develop tolerance when beingadministered as already described hereinabove.

Moreover, the present invention relates to a method for the productionof a vaccine composition comprising the step of combining a cell lysateobtainable by the process according to the present invention with anadjuvant or with dendritic cells.

The present invention also relates to a method for the production of apharmaceutical composition comprising the step of combining a celllysate obtainable by the process according to the present invention witha pharmaceutically acceptable carrier.

In another aspect the present invention relates to a method for thetreatment or prevention, e.g. by vaccination, of cancer, tumorousdiseases, infections and/or autoimmune diseases in an individualcomprising the step of administering to the individual a therapeuticallyor prophylactically effective amount of the lysate obtainable by theprocess according to the invention.

In the context of the present invention the term “individual” means asubject in need of a treatment or prevention of cancer, tumorousdiseases, infections and/or autoimmune diseases. Preferably, the subjectis a vertebrate, even more preferred a mammal, particularly preferred ahuman.

The term “administered” means administration of a therapeutically orprophylactically effective dose of the cell lysate of the invention toan individual. By “therapeutically or prophylactically effective amount”is meant a dose that produces the effects for which it is administered.The exact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art and described above, adjustments for systemic versuslocalized delivery, age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

In accordance with the present invention the term “vaccination” isrelated to a general process for immunization against cancers, tumorousdiseases, infections and/or autoimmune diseases. Vaccination is a formof deliberate artificial immunization whereby the cell lysates or withcell lysate loaded dendritic cells of the present invention areadministered. The cell lysates are administered in a form as describedherein, supra, and may sensitise the immune system such that if cancer,tumorous diseases, infections and/or autoimmune diseases arise withinthe body are being treated or prevented. See, for example, Immunology,at pp. 87-88; AMA Encyclopedia of Medicine at 573-574 and 1034; S. J.Cryz, Jr., in Immunotherapy and Vaccines, edited by Stanley J. Cryz, pp.3-11, VCH, Weihheim, Germany 1991. For an overview of the immune systemfrom a molecular perspective, see, for example, Mary S. Leffell, AnOverview of the Immune System: The Molecular Basis for Immune Responses,in Human Immunology Handbook pp. 1-45. Vaccination is also associatedwith immunization.

Immunization is a general term, and the term vaccination is used whenpatients are immunized. In general, immunization can be used as apreventive or as a therapeutic treatment. The preventive use ofimmunization is a prophylactic treatment, whereas the use ofimmunization while the disease is in progress is immunotherapy.Immunization provides two types of acquired immunity, active andpassive. Immunotherapy is the treatment of a disease by immunization,active or passive, or by the use of agents that modify the actions oflymphocytes. In particular, immunotherapy refers to the stimulation ofthe immune system and conventionally uses a form of immunostimulant, asubstance that causes a general, non-specific, stimulation of the immunesystem. The American Medical Association. Encyclopedia of Medicine, p.576 (this encyclopedia will hereinafter be referred to as “AMAEncyclopedia of Medicine”).

In a method for inducing an immune response to treat or prevent cancer,tumorous diseases, infections and/or autoimmune diseases, one or morecell lysates or with cell lysate loaded dendritic cells according to theinvention are provided, and an effective amount of one or more of thecell lysates or with cell lysate loaded dendritic cells are injected atleast once so as to permit release of biologically active quantities ofthe immunostimulant over a period of time to induce an immune responseto the presence of active tumor cells.

An individual for the purposes of the present invention includes bothhumans and other animals, preferably vertebrates and more preferablymammals. Thus the methods are applicable to both human therapy andveterinary applications. In a preferred embodiment the individual is amammal, e.g. a mouse, and in a most preferred embodiment the individualis human.

The compounds described herein having the desired therapeutic orprophylactic activity may be administered in a physiologicallyacceptable carrier to a patient, as described herein. Depending upon themanner of introduction, the compounds may be formulated in a variety ofways as discussed below. The concentration of therapeutically activecompound in the formulation may vary from about 0.1-100 wt %. However,it is also envisaged that the person skilled in the art is readily in aposition to determine the concentration of the therapeutically activecompound in the formulation by using his common general knowledge. Theagents maybe administered alone or in combination with other treatments,i.e., radiation, or other chemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater-soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The administration of the candidate agents of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intranodally, peritumorally, intratumorally,intrarectally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the candidate agents may bedirectly applied as a solution dry spray.

The cell lysates that are obtainable by the aforementioned processes canbe administered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical,intradermal, intranasal, intranodal, intrarectal, peritumotal,intratumoral or intrabronchial administration. The attending physicianand clinical factors will determine the dosage regimen. As is well knownin the medical arts, dosages for any one patient depends upon manyfactors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. A typical dose of lysate can origin from about 1000 to10¹³ cells and the typical dose for dendritic cells is about 10⁴ to 10¹²cells, however, doses below and above this exemplary range areenvisaged. Preferably, the dose of lysate corresponds to amountsgenerated from cells numbers between 10⁴ to 10¹² cells, more preferablybetween 10⁵ to 10¹¹ cells, more preferably between 10⁶ to 10¹⁰ cells.Preferably, the dose for loaded dendritic cells is between 10⁵ to 10¹¹cells, more preferably between 10⁶ to 10⁹ cells. Doses can vary betweenindividuals and can be split to multiple injections at different sitesand/or administration routes. Suitable and optimal doses can bedetermined by those skilled in the art. The amount of cell lysate usedfor loading dendritic cells can be determined by those skilled in theart, for example by those in vitro and or in vivo assays which areexemplary shown in the examples. Preferable amounts for lysatesoriginate from 10³ to 10¹³ cells, more preamble from 10⁴ to 10¹² cells,more preferable from 10⁵ to 10¹¹ cells, and more preferably from 10⁶ to10¹⁰ cells. Generally, the regimen as a regular administration of thepharmaceutical or vaccine composition should be in the range of 0,1 μgto 10 g per dose for the lysates, preferably 50 to 100 mg, amounts forfractionated lysates can be correspondently lower but may reach the highamounts. The dosages are preferably given once a week, however, duringprogression of the treatment the dosages can be given in much longertime intervals and in need can be given in much shorter time intervals,e.g., daily. In a preferred case the immune response is monitored usingherein described methods and further methods known to those skilled inthe art and dosages are optimized, e.g., in time, amount and/orcomposition.

If the regimen is a continuous infusion, it should also be in the rangeof 0.1 μg to 10 mg per kilogram of body weight per minute, respectively.Progress can be monitored by periodic assessment. The cell lysates ofthe invention may be administered locally or systemically.Administration will preferably be parenterally, e.g., intravenously,intranodally, intra peritoneally, intra tumourally, peri tumorally.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

It is also envisaged that the cell lysates are employed in co-therapyapproaches, i.e. in co-administration with other medicaments or drugs,for example anti-cancer drugs.

When vaccine therapy is carried out using the cell lysates or with celllysate loaded dendritic cells of the present invention, they may beadministered only once. However, it is desirable to repeat theadministration to the same site of a body to achieve coexistence of atumor antigen and a cytokine or a cytokine-inducing agent as long aspossible. For example, both components may preferably coexist for 3hours or more so that inflammatory reaction at the site ofadministration can be induced and conditions can be achieved whereinimmune cells are concentrated and cells are kept at the site. When acell lysate without adjuvant is administered, an adjuvant may beadministered to the same site. Generally, the cell lysate can beadministered to a patient from which the tumor material is derived;however, the vaccine can also be administered to a patient bearing atumor that contains, from a viewpoint of pathological diagnosis, thesame or relative species of a tumor antigen as that contained in thetumor material. The site to be administered is not particularly limited.Preferred sites include those where cytokines are hardly be diffused anddisappeared, for example, intradermal, subcutaneous or intramuscularsites, in lymphnodes, and in a main organ such as spleen. However, bychoosing a dosage form, which prevents ready diffusion of the activeingredients of the tumor vaccine, local administrations may sometimes beperformable to any site of a body, or by applying a drug deliverysystem, the systemic administration may sometimes be possible. The doseand administration period of the tumor vaccine of the present inventionare not particularly limited. It is desirable to determine anappropriate dose and administration period by observing effects of thevaccine therapy. The administration can be made, for example, byinjections and the like.

It is also preferred that the cell lysates or with cell lysate loadeddendritic cells are administered to an individual for the treatment orprevention of against infections, e.g. caused by microorganisms likebacteria, fungi, viruses and/or parasites or autoimmune diseases. Anautoimmune disease may arise from immune recognition and reactionagainst the individual's own cells or parts of the own body. Anotherpreferred embodiment for vaccination is a combination of an immunizationwith dendritic cells loaded a cell lysate according to the presentinvention followed by boosting with the necrotic cell lysate.

According to the present invention, the tumor cell lysates, which areused for the preparation of a pharmaceutical or vaccine composition, caneither be autologous or allogeneic or xenogenic with respect to thetreated individual. It is also envisaged that the tumor cell lysates canbe obtained from a tumor/tumor cell material/metastasis and used for thepreparation of a pharmaceutical or vaccine composition or cell lines,including cell lines, e.g., tumor cell lines, or primary cell lines orcell lines or cultures from primary material and the like administeredfor treating or preventing another kind of tumor. For example tumorcells derived from leukaemias or lymphomas can be used to treat orprevent colon carcinoma. In another preferred embodiment of the presentinvention the pharmaceutical or vaccine composition used for thevaccination comprises one or more tumor cell lysates produced fromdifferent tumors/tumor material/tumor cells in order to avoid aso-called tumor escape. These lysates can be loaded to dendritic cellsor their precursor states as described above and used as such as acomponent of vaccine or pharmaceutical compositions for administration.

Additionally, it is preferred that infected tumor cells as describedhereinabove are used for vaccination against infections, whereby theinfectious component harboured by the infected cells may be additionallyinactivated by chemical or physical means. It is also envisaged thatnon-infectious variants or mutants of the infectious component areneeded for infecting said tumor cells.

The invention also relates to the use of the lysate obtainable by theprocess according to the invention for the preparation of a vaccine orpharmaceutical composition for the treatment or prevention of cancer,tumorous diseases, infections and/or autoimmune diseases.

In a preferred embodiment the cancer or tumorous disease to be treatedor prevented is a cancer/tumorous disease of the head and neck, lung,mediastinum, gastrointestinal tract, genitourinary system,gynaecological system, breast, endocrine system, skin, childhood,unknown primary site or metastatic cancer, a sarcoma of the soft tissueand bone, a mesothelioma, a melanoma, a neoplasm of the central nervoussystem, a lymphoma, a leukaemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer. However, said cancer/tumorous disease may also beselected from those mentioned hereinabove in connection with the processaccording to the invention.

The infection to be treated or prevented is preferably bacterialinfection, viral infection, fungal infection, protozoal infection and/orhelminthic infection. More preferably the bacterial infection, viralinfection, fungal infection, protozoal infection and/or helminthicinfection is selected from the group consisting of bacterial infectionslike sepsis and sepsis shock, fever of unknown origin, infectiveendocarditis, intraabdominal infections and abscesses, acute infectious,diarrheal diseases and bacterial food poisoning, sexually transmitteddiseases, pelvic inflammatory disease, urinary tract infections andpyelonephritis, osteomyelitis, infections of the skin, muscle, and softtissues, infections in injection drug users, Infections from bites,scratches, and burns, infections in transplant recipients andhospital-acquired and intravascular device-related infections. Morepreferably the infection to be treated or prevented is selected from thegroup consisting of bacterial infections like pneumococcal infections,staphylococcal infections, streptococcal and enterococcal infections,diphtheria, other corynebacterial infections and anthrax, listeriamonocytogenes infections, clostridial infections, like tetanus,botulism, gas gangrene, antibiotic-assiociated colits, meningococcalinfections, gonococcal infections, moraxella (branhamella) catarrhalis,other moraxella species, and kingella infections, Haemophilus infectionscaused by haemophilus species, the HACEK group and other gram-negativebacilli infections, legionella infections, pertussis infections,gram-negative enteric bacilli infections, helicobacter infections,pseudomonas species and related organisms infections, salmonellainfections, shigella infections, campylobacter and related speciesinfections, cholera and other vibrio infections, brucella infections,tularaemia infections, plague and other yersinia infections, bartonellainfections, including cat-scratch disease, donovanosis (GranulomaInguinale) infections, nocardiosis, actinomycosis, infections due tomixed anaerobic organism, tuberculosis, leprosy (Hanses's Disease),infections due to nontuberculous mycobacteria, syphilis, endemictreponematoses, leptospirosis, relapsing fever, lyme borreliosis,rickettsial diseases, mycoplasma infections, chlamydial infections,viral infections due to Herpes simple viruses, Varicella-zoster virusinfections, Epstein-barr virus infections, including infectiousmononucleosis, Cytomegalovirus and human herpesvirus types 6, 7, and 8infections, smallpox, vaccinia, and other poxviruses infections,parvovirus inactions, Human papillomavirus infections, common viralrespiratory infections, influenzy, viral gastroenteritis, enterovirusesand reoviruses infections, measles, rubelly (German measles), mumps,rabies virus and other rhabdoviruses infections, infections caused byarthropod- and rodent-borne viruses, Marburg and ebola viruses(Filoviridae), fungal infections like histoplasmosis,coccidioidomycosis, blastomycosis, cryptococcosis, candidiasis,aspergillosis, mucormycosis, miscellaneous mycoses and protothecainfections, Pneumocystic carinii infection, protozoal infections likeamebiasis and infection with free-living amebas, malaria and otherdiseases caused by red blood cell parasites, leishmaniasis,trypanosomiasis, Toxoplasma infection, protozoal intestinal infectionsand trichomoniasis, heiminthic infections, like trichinosis andinfections with other tissue nematodes, Intestinal nematodes, filariasisand related infections (Loiasis, Onchocerciasis, and Dracunculiasis),schistosomiasis and other trematode infections or Cestodes infections.

The autoimmune disease to be treated or prevented is preferably selectedfrom the group consisting of allergic encephalomyelitis, autoimmunehaemolytic anemia, autoimmune thyroiditis, Hashimoto's disease,autoimmune male infertility, bullous pemphigoid, Celiac disease, Grave'sdisease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura,insulin-resistant diabetis mellitus, myasthenia gravis, perniciousanemia, pemphigus vulgaris, polyarteritis nodosa, primary biliarycirrhosis, Reiter's disease, rheumatic fever, sarcoidosis, Sjogren'sdisease, systemic lupus erythematosus, sympathetic ophthalmia, multiplesclerosis and/or viral myocarditis by Cocksakie B virus response.

In view of the in vivo and in vitro results of the examples of thepresent invention it is expected that the invention provides anadvantageous cancer, tumorous disease, infection and/or autoimmunedisease vaccine. In particular, mice developed both a humoral andcellular immune response when challenged with tumor antigens presentin/on tumor cells, which served as a source for the produced celllysate. Moreover, even antibodies against carbohydrate antigens havebeen developed which has not or rarely been observed so far whenconventionally produced tumor cell lysates have been administered.Additionally, it was found that mice with an implemented human immunesystem showed the same phenomenon.

The Figures show:

FIG. 1 Analysis of propidium iodide and annexin V-FITC labelledtemperature induced NM-F9 tumor cells (A-C) and anti-Hsp70 labeled NM-F9tumor cells by flow cytometry.

FIG. 2 Detection of cellular Hsp 70 expression in temperature treatedNM-F9 tumour cells derived from K562 tumor cells by immunocytochemicalstaining; panel a) shows NM-F9 cells incubated at 41.2° C., the stainingof Hsp70 positive cells was done by using a secondary Cy3-labelledantibody; panel b) shows NM-F9 cells incubated at 46.2° C. in the darkfield control; panel c) shows the same NM-F9 cells incubated at 46.2°C., wherein Hsp70 positive cells are labeled with a secondaryCy3-labelld antibody

FIG. 3 In vitro analysis of T cell (CD8) stimulation by temperaturetreated NM-F9 tumor cell lysate loaded dendritic cells

FIG. 4 In vitro analysis of T cell (CD4) activation by mature dendriticcells which were incubated with different NM-F9 tumor lysates.

FIG. 5 In vitro induction of CD8+ T-cell responses with various necroticNM-F9 cell lysates after one stimulation.

The tables show: TABLE 1 Induction of apoptosis, necrosis and expressionof membrane bound HSP 70 protein on NM-F9 tumor cells derived from K562by temperature treatment. Percent of total (%) Apoptotic Necrotic Hsp 70temperature cells cells cells positive cells 37.0° C. 70 +/− 3.7 21 +/−1.9 9 +/− 3.2 5 38.0° C. 64 25  9 1 40.0° C. 48 40 12 3 41.2° C. 44 4015 33 42.0° C. 34 45 20 1 42.4° C. 25 +/− 10  59 +/− 11  16 +/− 0.35 143.0° C. 34 47 19 0.7 44.0° C. 25 52 23 10 46.2° C.  5 +/− 0.6  9 +/−0.2 86 +/− 0.7  5

TABLE 2 Humoral immune response in NMRI mice induced by temperature pre-treated NM-F9 tumor cell lysates. Number of mice with antigen specificIgG immune response/total number Vaccine AGP TF Tn MUC1 Necrotic tumorcell lysate 46.2° C. 2/3 3/3 3/3 3/3 Apoptotic tumor cell lysate 42.4°C. 2/3 0/3 0/3 1/3 Lysate of tumor cells with 0/3 0/3 0/3 0/3 increasedHSP70 expression 41.2° C. Lysate of untreated tumor cells 37° C. 2/3 0/30/3 0/3

TABLE 3 Humoral immune response in NOD/SCID mice induced by temperaturepre-treated NM-F9 tumor cells lysates. Number of mice with antigenspecific IgG immune response/total number Vaccine AGP TF Tn MUC1Necrotic tumor cell lysate 46.2° C. 3/3 3/3 3/3 3/3 Apoptotic tumor celllysate 42.4° C. 1/3 1/3 1/3 0/3 Lysate of tumor cells with 0/3 0/3 0/30/3 increased HSP 70 expression 41.2° C. Lysate of untreated cells 37°C. 0/3 1/3 1/3 0/3 Negative control, Mel624 cell 0/3 0/3 0/3 0/3 lysate,AGP-, TF-, Tn-, MUC1-The examples illustrate the invention.1. Process of Temperature Treatment for Preparation of Apoptotic andNecrotic Tumor Cell Lysate

In the following Example K562 cells (ATCC: CCL-243) may be used.However, preferably TF-positive F9 cells (NM-F9) derived from K562 tumorcells (ATCC: CCL-243) are used which were cultivated in RPMI media with10% FCS, 1% glutamic acid (complete RPMI media), 8% CO₂, 95% humidity at37° C. For each preparation 7.5×10⁵ K562-cells were harvested. Beforetemperature induction tumor cells were resuspended in 450 microlitercomplete RPMI media (for mouse vaccination, section 5) or in serum-freeAIMV-media (for in vitro assay, section 4). For temperature inductionfour aliquots of 100 microliter and one aliquot of 50 microliter (0.5 mle-cups) of the tumor cells were incubated for 2 h in a preheatedthermocycler (Eppendorf, Hamburg, Germany). Afterwards temperaturetreated cells were resuspended in 25 ml complete RPMI media or AIMVmedia and recultured under conditions above (in complete RPMI media at37° C.). For induction of apoptosis and necrosis an incubation time of22 h found to be better than 6 h and 14.5 h (results described insection 2, flow cytometry). Before lysis, tumor cells were washed withPBS (for mouse vaccination) or AIMV (for in vitro assay) three times.Afterwards cell counts were adjusted to 5×10⁶ cells/75 microliter PBS(for mouse vaccination) or 1×10⁶/500 microliter AIMV (for in vitroassay). Cell lysis was performed by freezing in liquid nitrogen andthawing at 37° C. (water bath) for three cycles (30 sec/phase in liquidnitrogen). Cells lysates were stored at 80° C.

To improve the immunogenity and to simplify the induction process forpreparing necrotic cells, the duration of temperature induction (2 h or3 h) as well as the reculture time at 37° C. (0 h, 3 h, 22 h) werevaried. In these experiments temperature induction occurred in PBS orserum-free AIMV media in a thermoblock at 46.5° C. Heat-treated cellswere recultured in the same volume and media in microcentrifuge tubes inwhich temperature induction had taken place. Necrotic cell lysate (inAIMV-media) was used for in vitro analysis (FIG. 5). The followingpreparations were lysed by freezing and thawing: 1) Temperatureinduction 2 h (sample A) and 3 h (sample B) without reculture time. 2)Temperature induction 2 h (sample C) and 3 h (sample D) and 3 hreculture time. 3) Temperature induction 2 h and 22 h reculture time inmicrocentrifuge tubes (sample E). 4) as control: Temperature induction 2h and 22 h reculture time resuspended media.

2. Determination of Vital, Apoptotic and Necrotic Cells by FlowCytometry

For determination of vital, apoptotic and necrotic cells the ApoptosisDetection Kit I (Pharmingen, Heidelberg, Germany) containing AnnexinV-FITC and Propidium iodide was used; Apoptotic cells were only stainedby Annexin V-FITC, necrotic cells were stained by Annexin V-FITC andPropidium iodide (PI). For analysis. 3×10⁵ heat-treated and reculturedK562 cells (ATCC: CCL-243) may be used. However, TF-positive F9 cellsderived from K562 tumor cells (NM-F9) ( ) were used which were harvestedand washed in PBS twice. Cell pellets were resuspended in 1× bindingbuffer (10 mM Hepes pH 7.4, 140 mM NaCl and 2.5 mM CaCl 2) and stainedwith 5 microliter Annexin V-FITC and 2 microliter PI. After 15 minincubation time at room temperature (in a dark place) samples werediluted in 400 microliter 1× binding buffer. For flow cytometricanalysis the flow cytometer Coulter Epics XL of Beckman-Coulter (Miami,USA) with Expo 32 ADC software (Beckman-Coulter) was used. Forinvestigation the following parameter were used:

“sideward scatter”: 807 volt, “gain” 5, forward scatter”: 26 volt “gain”1.

Annexin V: FL1-channel, 707 volt, “gain” 1, compensation FL1-FL3 0.4.

PI: FL3-channel, 729 volt, “gain” 1, compensation FL3-FL1 17.9.

Proportional determination of vital, necrotic and apoptotic cells ofheat-treated 22 h recultured TF-positive F9 cells derived from K562cells (NM-F9) are described in “dot plots” diagrams in FIG. 1.K562-tumor cells were incubated in a thermocycler for 2 h at 37° C.(control, FIG. 1A), at 42.4° C. (FIG. 1B) and at 46.2° C. (FIG. 1C).After temperature induction cells were recultured 22 h at 37° C.

In a “dot plot” diagram the fluorescence intensity of Annex in V-FITC/PIstained cells is described by the position of spots in the coordinatefield. The coordinate field is separated into quadrants, whose positionis determined by negative and positive control using of PI or AnnexinV-FITC stained cells. The unstained population in the left quadrantbelow represents the vital cells (no dye can enter the cell), thepopulation in the right quadrant below represents the apoptotic cells(only Annexin V-FITC can enter the cell) and the population in the upperleft and right quadrant represents the necrotic cells.

To determine the best temperature for induction of apoptosis andnecrosis, TF-positive F9 cells derived from K562-cells were incubatedfor 2 h at 10 different temperatures described in table 1. As shown inFIGS. 1B and C the optimal temperature for induction of apoptotic cellsis 42.4° C. and the temperature for induction of necrotic cells is 46.2°C.

3. Preparation of Tumor Cell Lysate with High Expression of MembraneBound HSP 70 Proteins

In the following Example K562 cells (ATCC: CCL-243) may be used.However, TF-positive F9 cells derived from K562 (NM-F9) are preferablyused. With the latter cells it was observed that the preparation ofheat-treated TF-positive F9 cells derived from K562 (NM-F9) tumor celllysate with high expression of membrane bound HSP 70 is comparable tothe preparation of necrotic and apoptotic cells. The optimal reculturetime for TF-positive F9 cells derived from K562 cells (NM-F9) expressinghigh amounts of HSP 70 were 14.5 h (results, section 4).

Detection of Membrane Bound HSP 70 Protein by Flow Cytometry

3×10⁵ TF-positive F9 cells derived from K562 cells (NM-F9) which arepreferably used were harvested and resuspended in 25 microliter completeRPMI media. After 45 min incubation with anti-human HSP 70-IgG1 antibody(1:100 diluted in complete RPMI) at 4° C., cells were washed in PBS/10%FCS twice (5 min centrifugation at 1500 upm, Heraeus Multifuge).Afterwards the cells were incubated for 30 min with a secondaryCy3-anti-mouse-IgG antibody at 4° C. (1:200 diluted in PBS, Dianova,Hamburg, Germany). The cells were washed in PBS twice and resuspended in200 microliter PBS. For analysis the following-parameters were used:“sideward scatter” and “forward scatter” as already-described.

Cy3: FL2-chanel, 740 volt, “gain” 1.

The results are described in the overlay-histogram (FIG. 1 b), whichshow HSP 70 induction of the control (incubated at 37° C.) and F9cellsincubated at 41.2° C. As seen in the histogram in about 330% of theF9cell population incubated at 41.2° C. the expression of HSP 70proteins is increased.

The HSP 70 expression of TF-positive F9 cells derived from K562 cells(NM-F9) incubated for 2 h at 10 different temperatures is described intable 1. The optimal HSP 70 expression in heat-treated TF-positive F9cells derived from K562 cells (NM-F9) is received by temperatureinduction at 41.2° C. and 14.5 h recultured at 37° C.

Detection of Cellular HSP 70 Expression by Immunocytochemistry

The staining process occurred in a humid chamber.

For coating the slide 5×10⁴ cells in 50 microliter were dropped on theslide (in a humid chamber), 30 min incubated at 37° C. in the CO₂incubator and 1 h incubated at room temperature. After removing thesupernatant the samples were dried for 15 min at room temperature andstored at −20° C. wrapped in aluminium foil. After thawing the cellswere fixed in 5% formaldehyde (diluted in PBS, 5 min incubated at roomtemperature). The cells were washed in PBS three times and blocked byincubating 1 h with 5% BSA/PBS. After washing in PBS cells were labelledby anti-HSP 70 antibody (as above, 1:200 diluted in 1% BSA/PBS, 90 minincubation at RT). After washing the cells with PBS three times K562cells were incubated for 1 h with secondary Cy3-anti-mouse IgG antibody(1:200 diluted in 1% BSA/PBS) at room temperature. Finally the cellswere embedded in Mowiol-solution (6 g glycerin, 2.4 g mowiol 4-88(Calbiochem, Bad Soden, Germany), 6 ml H₂O, 12 ml 0.2 M Tris-HCl pH 8.5mixing 2 h at RT, 15 min centrifugation at 5000×g, addition ofDiazobizyclooctan (Sigma)). Immunochemical analysis was performed with aby Zeiss Microskope Axioplan (Oberkochen, Germany). The results ofcellular HSP 70—expression by F9 incubated at 41.2° C., incubated at42.4° C. (apoptotic cells) and incubated at 46.2° C. (necrotic cells) isshown in FIG. 2. 80-90% of K562 cells incubated at 41.2° C. haveincreased HSP 70 expression. In contrast 2-5% of necrotic cellsincubated at 46.2° C. showed increased HSP 70 expression.

For immunogenity studies lysates of four differently treated cellpopulations were prepared.

1) lysate of necrotic TF-positive F9 cells derived from K562 cells(NM-F9) (2 h temperature induction at 46.2° C., 22 h recultured at 37°C.), 2) lysate of apoptotic TF-positive F9 cells derived from K562 cells(2 h temperature induction at 42.4° C., 22 h recultured at 37° C.), 3)lysate of TF-positive F9 cells derived from K562 cells (NM-F9) withincreased expression of HSP 70, (2 h temperature induction at 41.2° C.,14.5 h recultured at 37° C.), 4) lysate of untreated TF-positive F9cells derived from K562 cells (incubated at 37° C.).

4. Specific Activation of Cb8+ and CD4+ T-Cells by Heat Treated TumorCell Lysate Taken Up, Processed and Presented by Dendritic Cells

Dendritic cells can be incubated with K562 cells, however, arepreferably incubated with different TF-positive F9 cells (NM-F9) derivedfrom K562 tumor lysates overnight. After phagocytosis of TF-positive F9cells derived from K562 lysates the maturation of dendritic cells wasinduced. Afterwards the activation of CD8+ (FIG. 3) and CD4+ (FIG. 4)T-cells by mature dendritic cells was analysed and the dendritic cellswere incubated with CD8+ and in order to stimulate these cells in anantigene-specific manner.

Preparation of Immature Human Dendritic Cells

Immature human dendritic cells were prepared by differentiation ofhuman-monocytes (hmoDC) by the method of Romani (Romani N et al. 1994,J. Exp. Med. 180: 83-93). Peripheral blood monocytes were isolated fromperipheral blood of healthy human donor by Ficoll gradientcentrifugation. Adherent cells which adhere on plastic were cultured for6 days in RPMI-1640, 10% FCS, 1000 U/ml GM-CSF 2.5 ng/ml TNFα and 1000U/m IL-4.

Secondly immature dendritic cells were prepared (Nemod-iDC, which areoptimized MUTZ-3 derived immature dendritic cells as described in WO03/023023 which can be purchased from NEMOD Immuntherapie AG,Robert-Rössle-Strasse 10, D-13125 Berlin, Germany)); from a human cellline according to the methods described in WO 03/023023

Loading and Maturation of Nemod-iDC

The immature hmoDC or Nemod-iDC. (10⁶ cells/sample) were incubatedovernight

with tumor cell lysates at a proportion of 1:1. After washing thedendritic cells with sterile PBS GM-CSF, IL-4 and 75 ng/ml TNFα wereadded. The mature hmoDC, Nemod-mDC became CD14−, CD1a+, CD80hi, CD86hi,CD40hi, MHCIIhi, CD83hi, DC-Sign+ (flow cytometry, suffix: −=noexpression, hi=high expression, +=positive expression). Prior to T cellsensitisation, the antigen loaded Nemod-mDC were irradiated with 30 Gy.

Preparation of Peripheral Blood T-Cells

T cells were isolated from the non-adherent fraction of PBMC of healthyHLA-A2 positive donor by a column of nylon wool (Polysciences Inc.,Eppelheim, Ger). Alternativaly CD4+ or CD8+-T-cells were isolated fromPBMC by CD4- or CD8-T-cell-MACS-Isolationkits (Miltenyi Biotec, Köln,Ger).

Induction of Tumor Cell Lysate Specific T-Cells

Total T-cells, CD4+ or CD8+ T-cells were incubated in serum-free media(AIM-V medium) with mature hmoDC or Nemod-mDC loaded with cell lysate inserum. The ratio of responder: stimulator (T-cell: DC) was 10-20:1.After overnight incubation 10 U/ml IL-2, 1.5 U/ml IL-1β and 5 ng/ml IL-7were added. After incubation for four days T-cells were restimulated bymature hmoDC or Nemod-mDC loaded with antigen. T-cells were analysed byIFNγ-ELISPOT Assay (FIGS. 3 and 5) following overnight incubation orafter 4 days (without addition of cytokine) the cell proliferation wasanalysed by BrdU assay (FIG. 4).

IFNγ-ELISPOT Assay

ELISPOT analysis was performed using a kit (Mabtech, Nacka, Sweden) andPVDF-bottomed 96-well-Multiscreen plates from Millipore (Bedford, USA).Before coating overnight with mouse-anti-human-IFNγ antibodies (15microgram/ml, at 4° C.) PVDF was soaked with 70% ethanol (50microliter/well). After washing with PBS, 7×10⁴ T-cells together withthe relevant number of antigen-loaded mature hmoDC or Nemod-mDC in 200microliter medium/well were incubated 16 h at 37° C. Cells were thenremoved and plates washed three times with PBS/Tween. Secreted IFNγ wasdetected by incubating with biotinylated anti-human-IFNγ antibody (50microliter/well in PBS, 2 h, RT), followed by a conjugate ofstreptavidin alkaline phosphatase (1:1000 dilution, 100 microliter/well,1 h incubation at RT). After washing with PBS-Tween four times,detection of alkaline phosphatase was achieved by staining with BCIP (35microliter/10 ml detection buffer) and NBT (45 microliter/10 mldetection buffer). The reaction was stopped by water. Prior to analysiswith an ELISPOT Reader (Autoimmun Diagnostika GmbH, Strassberg, Germany)96-well plates were dried for 1 h at 40° C.

T-Cell-Proliferation Assay (BrdU Incorparation)

BrdU was incorporated into proliferating T cells in accordance with themanufacture's protocol. After fixing, the cells were incubated with PODlabelled anti-BrdU-antibody. The subsequent staining reaction wasstopped by 1M sulfuric acid. Detection of antibody labelling wasachieved by photometry at an optical density of 450 nm (Ref. 690 nm).

Results of In Vitro Analysis

In vitro experiments revealed that the immunogenity of tumor celllysates was improved by heat pretreatment (FIGS. 3, 4 and 5). The bestresults were achieved by temperature induction at 46.2° C. (about 80%necrotic cells). This result was unexpected. In contrast to many authorspreparing necrotic cells by lysing untreated cells (Gallucci, S. NatureMedicine 1999; Kotera, Y. Cancer Res. 2001; Restifo, N.P. Curr. Opin.Immunol. 2000; Sauter, B. J. et al. Exp. Med. 2000) we improved theimmunogenity by temperature-induced necrosis of cells prior to lysis. Indisagreement with other authors (Dressel (2000), loc. cit; Feng (2001),loc cit.; Melcher (1998), loc. cit; Todryk (2000), loc. cit.) we couldnot improve the immunogenity by increasing the expression of HSP 70proteins (temperature induction at 41.2° C.) (FIGS. 3 and 4). Asdescribed in table I HSP 70 expression as determined by flow cytometry,was increased by temperature induction at 41.2° C. In both heat-inducednecrotic cells (46.2° C.) and apoptotic cells (42.4° C.) the surface HSP70 expression was reduced (necrotic cells) or unchanged (apoptoticcells) (table 1). These results were confirmed by immunofluorescencetests (FIG. 2, 2-5% of necrotic cells (46.2° C.) HSP 70 positive, 80-90%of cells incubated at 41.2° C. HSP 70 positive).

The highest cellular immunogenity (cytotoxic CD8+ T cells and CD4+ Thelper cells) was achieved by temperature-induced necrotic cell lysates.Compared to lysates of untreated cells, cell lysates of temperatureinduced apoptotic cells also induced an improved CD8+ T cell immuneresponse (FIG. 3). An improved cellular immune response was induced inthe autologous system (hmoDC) as well as in the semi-allogeneic system(NemodDC). A strong immune response is based on CD8+ and CD4+ T cellactivation. Therefore, development of tumor vaccines based on tumor celllysates can be improved by using temperature-induced necrotic celllysates. To simplify the process and to improve the immunogenity thepreparation process of temperature-induced necrotic cells was optimised.All necrotic TF-positive F9 cells derived from K562 tumor cells (NM-F9)induced by a variety of preparation process (mentioned in section 1)show high immunogenity in “in vitro assay” (FIG. 5). Necrotic celllysates induced by 2 h incubation at 46.5° C. without reculture (section1, sample A) induced a small increase in CD8+ T-cell reaction.

5. Vaccination of Mice by Temperature Treated Human Tumor Cell Lysine

a) “normal” NMRI Mice

NMRI mice were vaccinated subcutaneously with lysates oftemperature-treated tumor cells (5×10⁶ cells/mouse) of K562 derived F9cells (NM-F9). After two weeks mice were boosted with the same tumorvaccine. No tumor growth or other side effects were observed during thevaccination period. One day before immunisation and 9 and 27 days afterimmunisation mice were bled to analyse serum for. TF-, Tn-, MUC 1- andasialoglycophorin A antibodies by ELISA. For these analyses, 96-wellplates were coated withThomsen-Friedenreich-disaccharide-polyacrylamide-conjugate (TF-PAA),Tn-monosaccharide-PM-conjugate (Synthesome, Munich, Germany),asialoglycophorin A (AGPA, Sigma) (2 microgram/ml in 50 microliter PBS)and MUC 1 (diluted 1:40 in PBS, optimal dilution was determined byanti-MUC1-antibody A76-A/C7) purified from supernatant of tumor bells asdescribed in PCT/EP03/08014 (After incubation overnight at 4° C. 96-wellplates were washed with 0.05% Tween 20 in PBS (washing buffer), blockedwith 5% BSA, 0.05% Tween 20 in PBS (1.5 h incubation at RT) and washedagain in washing buffer three times. The coated 96-well plates wereincubated with different dilutions of mice sera for 2 h at roomtemperature. The purified antibodies A76-A/C7, A78-G/A7 (Cao (1997),Virchows Arch. 431, 159-166) and Tn-HB1 (diluted 1:500, Dako, Hamburg,Germany) were used as positive controls. For negative control theprimary antibody was replaced by medium. After washing in washing bufferthree times plates were incubated with peroxidase labelled anti-mouseIgG or IgM (isolated from rabbit, diluted 1:5000, Dianova). Finallyplates were washed with washing buffer twice and with PBS one time. TheELISA was developed with 0.4 mg/ml o-phenyldiamine (Sigma) in 25 mMcitrate-phosphate buffer pH 5.0 with 0.04% H₂O₂ at room temperature (inthe dark). The colour reaction was stopped by addition of 2.5 N sulfuricacid (final concentration 0.07 N) and analysed by ELISA-reader at 490 nm(reference filter at 630 nm)

b) Specific activation of human IgG antibody immune response in NOD/SCIDmouse model by vaccination of different temperature pre-treated tumorcell lysate

NOD-SCID mice provide a commonly used mouse model, popular because oftheir deficient immune system. In these mice the human immune system isestablished by intraperitoneal application of human peripheral bloodlymphocytes into mice irradiated one day earlier (PBL, standardpreparation, 5×10⁷ cell/mouse). 2-4 h after application of PBL, micewere vaccinated subcutaneously with tumor cell lysates of untreated,apoptotic, necrotic or HSP70 expressing cells as an emulsion withincomplete Freund's adjuvant (5×10⁶ cells/mouse, cells+adjuvant=100′microliter). After 14 days mice were boosted by the same cell lysates.Cell lysates of MeI624 cells (5×10⁶ cells/mouse) were used as negativecontrol. There was no evidence of tumor growth or other side effects inNOD-SCID mice. For analysis of serum, mice were bled at days 13 and 28after the first immunisation. The analyses were carried out by ELISA asdescribed above, here the dilution of secondary POD labelled anti humanIgG antibody was 1:10 000.

Results of In Vivo Analysis

A humoral IgG immune response against all tested antigens was induced innearly all NMRI mice and NOD-SCID mice vaccinated by temperature inducednecrotic cell lysate (tab. 2 and 3). An IgG immune response against TFand Tn carbohydrate antigen is unusual because immunisation of mice byTF-antigen usually induces an IgM immune response. This might indicatethat activation of the immune response by temperature-induced celllysates is superior immunisation process involving a switch of antibodyclass associated with a T helper cell immune response as well asinduction of memory immune responses against the above antigens. Aspecific human IgG immune response was induced in a murine immune systemas well as in an implantated human immune system. Clearly cell lysatesof necrotic cells induced a stronger specific humoral immune responsethan cell lysates of apoptotic cells. Cell lysates of tumor cells withincreased HSP 70 expression (temperature induction at 41.2° C.) did notinduced a specific humoral immune response in either mouse model (tab. 2and 3). These are not in accordance with results of other authors(Dressel (2000), loc cit.; Feng (2001), loc. Cit.; Melcher, (1998), loc.cit.; Todryk (2000), loc. cit.) who described a strong immune responseinduced by cells with increased HSP 70 expression. Cell lysates ofuntreated tumor cells induced only poor specific humoral immuneresponses.

1. A process for the production of an immunogenic compound comprisingthe steps of (a) inducing necrosis of tumor cells by subjecting thecells to a temperature of more than 41.2° C. for at least 15 minutes;and (b) lysing said necrotic tumor cells so as to obtain a lysate. 2.The process of claim 1, wherein necrosis is induced in tumor cellsselected from the group consisting of tumor cell lines, cells derivedfrom primary tumor material, cells derived from cell populations ofprimary tumor material and/or metastases including micrometatases. 3.The process of claim 1, wherein said induction of necrosis is achievedby incubating said tumor cells at a temperature of more than 42° C. 4.The process of claim 1, wherein said induction of necrosis is achievedby incubating said tumor cells at a temperature in the range of 45° C.to 55° C.
 5. The process of claim 1, wherein said induction of necrosisis achieved by incubating said tumor cells at a temperature in the rangeof 45.5° C. to 47° C.
 6. The process of claim 1, wherein said inductionof necrosis is performed in the range of 2 to 3 hours.
 7. The process ofclaim 1, wherein more than 40% of said tumor cells are necrotic afterinduction of necrosis.
 8. The process of claim 1, wherein more than 70%of said tumor cells are necrotic after induction of necrosis.
 9. Theprocess of claim 1, wherein said tumor cells are genetically engineered,mutated or infected by oncogenic viruses.
 10. The process of claim 1,wherein said tumor cells are autologous and from the same or fromdifferent tissues, organs or cell origin in a species.
 11. The processof claim 1, wherein said tumor cells are allogeneic.
 12. The process ofclaim 1, wherein said tumor cells are syngenic.
 13. The process of claim1, wherein said tumor cells are xenogenic.
 14. The process of claim 1,wherein more than one type of tumor cell is used and wherein the tumorcells are from the same or different individuals, tissues, cell types ortumors.
 15. The process of claim 1, wherein said tumor cells are NM-F9cells (DSMZ deposit No DSM ACCC2606) or NM-D4 cells (DSMZ deposit No.DSM ACC2605).
 16. A lysate obtainable by the process of claim
 1. 17.Dendritic cells loaded with the lysate of claim
 16. 18. A compositioncomprising a lysate of claim 16 or dendritic cells of claim
 17. 19. Thecomposition of claim 18, which is a pharmaceutical composition.
 20. Thecomposition of claim 18, which is a vaccine composition.
 21. Thepharmaceutical composition of claim 19, which is optionally combinedwith an adjuvant.
 22. The dendritic cells of claim 17, wherein saiddendritic cells are immature.
 23. The dendritic cells of claim 17,wherein said dendritic cells are mature.
 24. A method for the productionof a vaccine composition comprising the step of combining a cell lysateof claim 16 or the dendritic cells of claim 17 with an adjuvant.
 25. Amethod for the production of a pharmaceutical composition comprising thestep of combining a cell lysate of claim 16 with a pharmaceuticallyacceptable carrier.
 26. A method for the treatment or prevention ofcancer, tumorous diseases, infections and/or autoimmune diseases in apatient in need thereof comprising administering a therapeutically orprophylactically effective amount of the lysate of claim 16 or thedendritic cells of claim 17 to the patient.
 27. (canceled)
 28. Themethod of claim 26, wherein said cancer or tumorous disease is a cancerof the head and neck, lung, mediastinum, gastrointestinal tract,genitourinary system, gynaecological system, breast, endocrine system,skin, childhood, unknown primary site or metastatic cancer, a sarcoma ofthe soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of thecentral nervous system, a lymphoma, a leukaemia, a paraneoplasticsyndrome, a peritoneal carcinomastosis, a immunosuppression-relatedmalignancy and/or metastatic cancer.
 29. The method of claim 26, whereinsaid infection is bacterial infection, viral infection, fungalinfection, protozoal infection and/or helminthic infection.
 30. Themethod of claim 26, wherein said autoimmune disease is allergicencephalomyelitis, autoimmune haemolytic anemia, autoimmune thyroiditis,Hashimoto's disease, autoimmune mail infertility, bullous pemphigoid,Celiac disease, Grave's disease, Goodpasture's syndrome, idiopathicthrombocytopenic purpura, insulin-resistant diabetis mellitus,myasthenia gravis, pernicious anemia, pemphigus vulgaris, polyarteritisnodosa, primary biliary cirrhosis, Reiter's disease, rheumatic fever,sarcoidosis, Sjogren's disease, systemic lupus erythematosus,sympathetic ophthalmia, multiple sclerosis and/or viral myocarditis byCocksakie B virus response.
 31. The vaccine composition of claim 20,which is optionally combined with an adjuvant.
 32. The composition ofclaim 18, wherein the dendritic cells are immature.
 33. The compositionof claim 20, wherein the dendritic cells are mature.
 34. A method forthe treatment or prevention of cancer, tumorous diseases, infectionsand/or autoimmune diseases in a patient in need thereof comprisingadministering a therapeutically or prophylactically effective amount ofthe pharmaceutical composition of claim 19 to the patient.
 35. A methodfor the treatment or prevention of cancer, tumorous diseases, infectionsand/or autoimmune diseases in a patient in need thereof comprisingadministering a therapeutically or prophylactically effective amount ofthe vaccine composition of claim 20 to the patient.